Modulation of Taste Related Behavior by Peptide YY Signaling

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Modulation of Taste Related Behavior by Peptide YY Signaling
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Lasala, Michael S
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Master's ( M.S.)
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University of Florida
Degree Disciplines:
Medical Sciences, Medicine
Committee Chair:
Dotson, Cedrick Deshawn
Committee Members:
Zolotukhin, Serge
Ache, Barry W

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buds -- cell -- cytology -- fat -- hormones -- modulation -- npy -- oral -- peptideyy -- pyy -- receptor -- saliva -- taste
Medicine -- Dissertations, Academic -- UF
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Abstract:
Recent evidence reveals that Peptide YY (PYY), a peptide hormone expressed by cells in the gastrointestinal tract, is also synthesized and secreted by cells in the oral cavity. It now joins the list of the many metabolic polypeptides that are expressed in taste cells and/or present in saliva. This is one of many common characteristics that link the gastrointestinal and gustatory systems together. The current study sheds light on the locations of PYY and its cognate receptors in taste buds and how its signaling impacts taste perception. By immunofluorescent techniques, I found that PYY, NPY1R and NPY2R are all expressed in taste receptor cells. Using brief-access taste testing, I was able to determine that PYY signaling in the oral cavity modulates bitter and fat taste responsiveness. Using pharmacological techniques with Y receptor antagonists, I demonstrated that NPY1R mediates the responsiveness for bitter stimuli. I also demonstrated by viral vector therapy, that long term augmentation of peptide YY in saliva, rescues responsiveness to lipids, but not to bitter stimuli. Lastly, I found that the expression of NPY receptors is dependent on metabolic state suggesting that this system may be dynamically modulated. The current report has the unique attribute of addressing, for the first time, basic questions of PYY and Y receptor family biology as related to taste perception and tests the novel and emerging hypothesis that gastrointestinal hormonal systems, acting at the initial level of sensory transduction, profoundly influences gustatory behavior.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Michael S Lasala.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Dotson, Cedrick Deshawn.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-05-31

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1 THE MODULATION OF TASTE RELATED BEHAVIOR BY PYY SIGNALING By MICHAEL STEPHEN LA SALA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MAS TER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Michael La Sala

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3 To my parents, I am a p roduct of your love and support

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4 ACKNOWLEDGMENTS First and foremost I would like to thank my mentor Dr. Shawn Do tson. Shawn has given me all of the guidance a graduate student can ask for. He has helped me develop skills that I will carry for the rest of my life, as a young professional and scientist. His compassion for his students, our development and our future s uccess is unparalleled. He has been there for me even in my greatest times of need and I owe him all of the gratitude in the world. Along these lines I would like to thank Dr. Zolotukhin for taking me in as an undergrad and allowing me to work in his lab I would also like to thank the final member of my committee Dr. Barry Ache for his expert advice in this field. I am grateful to Dr. Daniela Hurtado for everything she has taught me during my techniques and protocols which allowed me to perform most of my experiments. She has always been there providing me with a helping hand and I am thankful to have had the privilege of working with such a great coworker and friend. I would like to thank othe r members of the Dotson and Zolotukhin lab including Alicia Brown, Seth Curlin, Dr. Damien Marsic, David Duncan, Dr. especially Ramaz Geguchadze along with various volunteers for their close support along the way especially Haseeb Khan and Myra Quiroga. I want to thank our collaborators Dr. Scott Herness and Tamara Kolli for their help with tissue preparation techniques, Martha Campbell Thomson for her help with staining techniques Oleg Gorbatyuk for confocal Finally I would like to thank my family for their love and support and my friends for making my graduate experience a most memorable one

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5 TAB LE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 The Gustatory System ................................ ................................ ............................ 14 Taste Perception ................................ ................................ .............................. 14 Taste Receptors ................................ ................................ ............................... 16 Signal Transduction Pathways ................................ ................................ ......... 18 Neurotransmitters and Neuromodulators ................................ .......................... 21 Hormone Signaling in Taste buds ................................ ................................ .... 23 Peptide YY and its Signaling in the Oral Cavity ................................ ...................... 29 2 CHARACTERIZING THE EXPRESSION OF PYY AND THE NPY RECEPTORS IN THE PERIPHERAL GUSTATORY SYSTEM ................................ ..................... 33 Materials and Methods ................................ ................................ ............................ 34 Animal Models and Tissue Collection ................................ ............................... 34 Immunofluorescence ................................ ................................ ........................ 35 Double Labeling Immunofluorescence ................................ ............................. 37 TSA Double labeling Technique ................................ ................................ ....... 37 Quantificati on of Immunoreactive Cells ................................ ............................ 38 Expression of Y2R following Acute Augmentation of Salivary PYY 3 36 ............ 39 Results ................................ ................................ ................................ .................... 40 Confirmation of PYY Localization in Taste Buds ................................ .............. 40 Distribution of GFP in Tissues of a PYY GFP Transgenic Mouse Model. ........ 41 Quantification of PYY Positive Taste Cells ................................ ....................... 42 Y1 Receptors are Primarily Expressed in Type II Taste Receptor Cells ........... 43 Effects of Salivary PYY 3 36 on the Expression of Apical Y2 Receptors in Taste Buds. ................................ ................................ ................................ ... 43 Discussion ................................ ................................ ................................ .............. 45 3 TASTE MODULATION BY PYY SIGNALING ................................ ......................... 60

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6 Materials and Methods ................................ ................................ ............................ 62 Animals ................................ ................................ ................................ ............. 62 Taste Stimuli ................................ ................................ ................................ ..... 62 Chronic Salivary Augmentation of PYY 3 36 ................................ ...................... 63 Brief access Taste Tests ................................ ................................ .................. 64 Statistical Analysis ................................ ................................ ............................ 64 Results ................................ ................................ ................................ .................... 65 PYY Signaling Modulates Fat and Bitter Taste Respons iveness in Mice. ........ 65 Impacts of Y1 Receptor on Taste Responsiveness. ................................ ......... 65 The Effects of Salivary PYY 3 36 on Fat Taste Perception. ................................ 66 Discussion ................................ ................................ ................................ .............. 67 4 CONCLUSIONS AND GENERAL DISCUSSION ................................ .................... 75 Charact erizing the Expression of PYY and the NPY Receptors in the Peripheral Gustatory System ................................ ................................ ................................ 76 Taste Modulation by PYY Signaling ................................ ................................ ........ 79 T heory of PYY Signaling in the Oral Cavity ................................ ............................ 81 LIST OF REFERENCES ................................ ................................ ............................... 85 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 94

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7 LIS T OF TABLES Table page 2 1 Percentages of GFP positive taste cells colocalized with cell markers.. ............. 57 2 2 Percentages of Y1R positive taste cells colocalized with cell markers. ............. 58 2 3 Antibodies used in Immunohistochemistry experiments ................................ ..... 59 2 4 Control experiments performed for Antibodies ................................ ................... 59

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8 LIST OF FIGURES Figure page 2 1 Positive PYY immunofluorescence i 2 positive type II taste cells.. ......... 50 2 2 GFP immunoreactivity throughout PYY GFP Transgenic Murine Tissue.. ......... 51 2 3 GFP double labeling immunofluorscence in CVP taste buds of PYY GFP transgenic. ................................ ................................ ................................ .......... 52 2 4 2 positive type II taste cells. ................ 53 2 5 L ocalization of Y2 receptor expression in apical taste pores of murine taste buds. ................................ ................................ ................................ ................... 54 2 6 Expression of Apical Y2 Receptor i n the Tast e Bud based on Metabolic State. ................................ ................................ ................................ .................. 55 2 7 T he effects of oral administration of PYY 3 36 on apical expression of Y2R in taste buds. ................................ ................................ ................................ .......... 56 3 1 Diminished responsiveness of PYY / mice to fat and bitter tasting stimuli. ........ 72 3 2 Y1 receptor mediated responses to taste stimuli. ................................ ............... 73 3 3 Salivary PYY 3 36 modulates fat taste perception. ................................ ................ 74 4 1 Theory of PYY Signaling in the Oral Cavity. PYY diffuses though leak y capillaries into cells of the salivary glands. ................................ ......................... 84

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9 LIST OF ABBREVIATIONS 5 HT Serotonin ACh Acetylcholine MSH Alpha Melanocyte stimulating H ormone ATP Adenosine Triphosphate Ca 2+ Calcium ion cAMP Cyclic Adenosine Mono phosphate. CCK Cholecystokinin CD36 Cluster of Differentiation 36 CVP Circumvallate Papilla DAPI 4',6' diamino 2 phenylindole2HCl DB Denatonium Benzoate Db/db Homozygous Diabetic Mouse ddH2O Double Distilled Water DMSO Dimethyl sulfoxide DPP IV Dipeptidy l Peptidase 4 ENaC Epithelium Sodium Channel GFP Green Fluorescent Protein GLP 1 Glucagon like Polypeptide 1 GlucR Glucagon Receptor GPCR G protein Coupled Receptor H + Hydrogen ion H2O2 Hydrogen Peroxide HCL Hydrochloric Acid IACUC Institutional Animal Car e and Use Committee

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10 IP 3 I nositol 1,4,5 trisphosphate K + Potassium ion Kir Inward rectifying K + ion channel KO Knockout LCFA Long Chain Fatty Acids MALDI TOF Matrix assisted laser desorption ionization time of flight mass spectrometry MCFA Middle Chain Fat ty Acids mM Milli Molar mRNA Messenger Ribonucleic Acid Na + Sodium ion NaCl Sodium Chloride NBF Neutral Buffered Formalin NCAM Neural Cell Adhesion Molecule NPY Neuropeptide Tyrosine; Neuropeptide Y Ob R Leptin Receptor OCT Optimum Cutting Temperature OS O ral Spray PBS Phosphate buffered S aline PKA Protein Kinase A PKDL Polycycstic Kidney Disease 2 Phospholipase C beta 2 POMC Pro opiomelanocortin PP Pancreatic Polypeptide PYY Peptid e Tyrosine Tyrosine; Peptide YY rAAV5 Recombinant Adeno Associated Viru s Serotype 5

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11 RP HPLC Reverse Phase High Performance Liquid Chromatography RT PCR Real Time Polymerase Chain Reaction S.E.M. Standard Error of the Mean SNAP Synaptosomal associated Protein. T1R Taste Receptor Type 1 T1R1 Taste Receptor Type 1 Member 1 T1R2 Taste Receptor Type 1 Member 2 T1R3 Taste Receptor Type 1 Member 3 T2R Taste Receptor Type 2 TNB Tris NaCl Blocking Buffer TNT Tris HCL NaCL Tween20 buffer TSA Tyramide Signal A mplification TRPM5 Transient Receptor Potential Cation Channel S ubfamily M memb er 5 TRPV1 Transient Receptor Potential Vanilloid 1 VIP Vasoactive Intestinal Peptide YR Neuropeptide Y Receptor (1, 2, 3, 4 or 5) m Micrometers M Micro Molar

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12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE INFLUENCE OF PEP TIDE YY SIGNALING ON TASTE RESPONSIVENESS By Michael Stephen La Sala May 2013 Chair: C. Shawn Dotson Major: Medical Sciences Recent evidence reveals that Peptide YY (PYY), a peptide hormone expressed by cells in the gastrointestinal tract, is also synthesized and secreted by cells in the oral cavity. It now joins the list of the many metabolic polypeptides that are expressed in taste cells and/ or present in saliva. This is one of many common characteristics that link the gastrointestinal and gustatory systems together. The current study sheds light on the locations of PYY and its cognate receptors in taste buds and how its signaling impacts tast e perception. By immunofluorescent techniques I found that PYY, NPY1R and NPY2R are all expressed in taste receptor cells. Using brief access taste testing, I was able to determine that PYY signaling in the oral cavity modulates bitter and fat taste respo nsiveness. Using pharmacological techniques with Y receptor antagonists I demonstrate d that the observed e ffect on taste related behavior of disrupting PYY signaling results from the loss of local signaling in the oral cavity and that, specifically, NPY1R likely mediates the observed behavior to bitter stimuli. Furthermore, by viral vector therapy, I demonstrated that long term augmentation of peptide YY in saliva rescues responsiveness to lipids, but not to bitter stimuli. Lastly, I found that the expres sion of NPY receptors is dependent on metabolic state suggesting that this

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13 system may be dynamically modulated. The current report has the unique attribute of addressing, for the first time, basic questions of PYY and Y receptor family biology as related t o taste perception and tests the novel and emerging hypothesis that gastrointestinal hormonal systems, acting at the initial level of sensory transduction, profoundly influence s gustatory behavior.

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14 CHAPTER 1 INTRODUCTION The gustatory system acts as a sen tinel allowing us to recognize external chemical stimuli entering the alimentary canal Together with receptors of the olfactory and somatosensory systems, gustatory receptors recognize distinct characteristics of many chemicals that comprise ingested food s. Receptors relay sensory signals to the brain which segregates, evaluates, and distinguishes the stimuli, leading to the [1] In recent history, it has been revealed that there are phenotypic characteristics shared by the gustatory and gastrointestinal system. For example, the intestinal epithelium harbors apical chemoreceptors on enteroendocrine cells that detect stimuli in the lumen of the intestinal tract, a similar event that occurs with taste cells in the oral cavity [2 3 4 5 ] Furthermore, it is also apparent that gastrointestinal hormones and the ir receptors are also expressed within the peripheral gustatory system [6 7] It has been suggested that these hormones may be playing a role in the modulation of taste perception. The focus of this chapter is to describe the peripheral gustatory system, emphasizing the role s that hormone signali ng plays in taste perception. The Gustat ory System Taste Perception The sense of taste begins with chemical stimuli that enter the body through the oral cavity. These stimuli give rise to specific taste qualities dependent upon the class of receptors that are activated on taste cells. To date, t here are five well characterized taste modalities that fit this description: sweet (simple carbohydrates), salty (electrolytes such as sodium), umami (amino acids), bitter (potentially toxic compounds such as

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15 some alkaloids found in plants) and sour (acids ). The gustatory systems primary function is to recognize properties of these taste modalities as being either appetitive (i.e. sweet low intensity salt, umami ) or aversive (i.e. bitter, high intensity salt, sour). Additionally, there is emerging evidence that lipids can be detected by fatty acid receptors on taste cells leading to the development of a sixth taste known as fat [8 12] Experiencing these taste qualities begins with specialized anatomical structures known as taste buds. In mammals, about 75% of taste buds are located on the dorsal surface of the tongue in structures called papillae [13] Eleven percent of taste buds appear in the soft palate mostly along a junction referred to as the G esc hmacksstreipen (taste strip) [13] The remaining taste buds can be found in the larynx, nasopharynx and epiglottis. There are four types of papillae on the tongue, three of which contain taste buds. The fungiform papillae are dispersed th roughout the anterior dorsal surface of the tongue harboring a single taste bud at the apex of the papillae The foliate papillae are composed of several deep trenches located posteriorly on lateral portions of the tongue. Finally, posterior of the inter molar eminence and in between the foliate trenches, lies the circumvallate papilla (CVP) containing the densest region o f taste buds in the oral cavity [14] Rodents generally have single CVP where as humans have several. Taste buds are comprised o f 50 150 taste cells per bud [15] These taste cells have been c ategorized into four cell types based on their cytological and ultrastructural features: type I, type II, type III and type IV [15] Type I cells (supporting cells) are electron dense, containing dark cy toplasms and indented nuclei [15] These taste cells are thought to play a role in supporting the structural integrity of the taste bud and the

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16 cells that surround it. They also have the potential to regulate the extracellular envir onment within the taste bud [16] While type I cells have not traditionally been recognized for the tra nsduction of taste stimuli, a recent report suggests that amiloride sensitive Na+ channels are located on type I cells implicating their involvement in salt taste transduction [17] Type II taste cells (taste receptor cells) have a translucent cytoplasm and contain a large round nucleus [15] They are referred to as taste r eceptor cells because they express G protein coupled receptors (GPCRs) and other receptors responsible for recognizing various taste stimuli. Expressed primarily in type II taste cells, GPCRs are responsible for the transduction of stimuli that give rise t o sweet, bitter, umami and f at taste precepts [8,12,18 24] Type III taste cells have round nuclei like type II cells but also have dark cytoplasm similar that of type I [15] T his cell type contains traditional synapses with gustatory nerve fibers, an d express various synaptic molecules as well as neurotransmitters [16] Finally, type IV cells are located on the basolateral part of taste buds and are comprised of progenitor cells. These cells contain sonic hedgehog genes which allow for t he differentiation and maturation into other taste cell types. Unlike the other cell types, t hese cells are not believed to be involved in the signal transduction of tastes [25] Taste R ece ptors Type II and type III taste cells extend their cell bodies and project through tight, claudin rich openings known as the taste pore. The se tight junctions prevent stim uli from entering the taste bud and restrict stimuli receptor interactions to the apical projections of taste cells. There are many families of receptors expressed on taste receptor cells that are responsible for recognizing taste stimuli that corresponds to each taste modality Sweet, bitter and uma mi tastes are mediated by GPCRs [20 23] Salt

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17 taste is regulated by apical ion channels ( e.g. ENaC) that allow the direct passage of Na + into the cell. S our (acids) detection is not well characterized, but possible mechanisms such as the diffusion of H + ions directly into the cell and /or H + detection by PKD1 like family receptors are currently being studied [1,26,27] Finally, as evidence about a sixth taste continue s to emerg e, lipids have now been shown to interact with various fatty acid receptors that are expressed in taste cells and are known to be involved in the transduction of fat taste [8,9,12,24,28,29] GPCRs are associated wit h intracellular G proteins that become secondary messengers for downstream cell signaling. A major advance in taste research was the discovery of T1R and T2R GPCRs in taste cells Receptors of the T1R family are responsible for recognizing sweet and umami taste stimuli. Specifically, T1R3 dimerizes with T1R2 to transduce sweet taste [20,21] For umami taste, T1R1 dimerizes with T1R3 to detect amino acids that comprise proteins [22,23] Another class of GPCRs, T2Rs, interacts with various ligands that give rise to bitter taste percepts [30] GPR40 and GPR120 are GPCRs that have been implicated in the orosensory perception of dietary lipids [9,12,24,29] In taste buds, both rece ptors vary in their location a s well as their preference for fatty acid chain length. GPR40 is primarily expressed in type I taste cells whereas GPR120 is mostly present in type II taste cells [9] Taste preference studies using GPR40 and GPR120 knockout mice show ed a reduced preference for LCFAs compared to wild type mice [9] Furthermore, the same study showed that GPR40 binds with similar affinity to middle and long chain fatty acids (MCFA, LCFA), while GPR120 binds with greater affinity to LCFAs [9] Together these

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18 data suggest that both GPR40 and GPR 120 are playing contributing roles in fat detection [9,24] A fatty acid taste receptor of the class B scavenger family, the cluster of differentiation 36 commonly known as CD36, has also be en discovered in taste cells [8] CD36 is a membrane bound g lycoprotein associated with the Src Kinase intracellu lar signal transduction pathway [8] By double labeling imm unohistochemistry, CD36 was found to be localized p rimarily in type II taste cells [8] CD36 knockout mice showed a diminished proclivity to detect LCFAs in comparison to wild type preference [8] O ne study showed that there is an increase in intracellular Ca 2+ induced by LCFA s. The effect was dependent u pon CD36 receptor regulation [31] Furthermore, up to three LCFAs can bind to CD36 with greater affinity than GP R120 or GPR40 [32] Since CD36 can bind a greater number of fatty acids with higher affinity compared to GPR120, it is suggested that CD36 plays the primary role for lipid detection in taste cells. However, the interaction between CD36 and GPR120 has yet to be studied. There is some data suggesting that both receptors are colocated on lipids rafts of the plasma membrane s as seen i n other parts of the b ody [10,33,34] In one instance, CD36 has been shown to act as a shuttling agent for lipids to other signal transducing receptors [35] If this is the case CD36 could be shut tling LCFAs to GPR120 for lipid detection. Other theories have arisen about the specific roles of CD36 and GPR120 in taste cells; h owever, further studies are needed to fully characterize their respective roles. Signal Transduction Pathways Upon activatio n of GPCRs, signal transduction pathways invol ving secondary messengers ensue Heterotrimeric G proteins, including the taste specific protein gustducin, dissociate from their receptors, uncouple into subunits and activate various

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19 downstream effectors. Ade nylate cyclase is responsible f or cyclic AMP (cAMP) production. It is thought that adenylate cyclase is activated by G proteins in response to [36] The increase in cAMP leads to the phosphorylation of protein kinase A (PKA). PKA then phosphorylates basola teral K + c hannels leading to the depolarization of the taste cell. As the cell becomes de polarized, voltage gated Ca 2+ channels open leading to an influx of Ca 2+ into the cell. The increase in Ca 2+ opens TRPM5 Na + channels allowing the passage of Na + into the cell Finally, the increase in intracellular Ca 2+ mobilizes secretory granules to exocytose neurotransmit ters at the basolateral portion of the taste cell Another transduction pathway implicated in sweet, bitter, umami and fat tastes involves the act 2) by G proteins [37] Activated by the beta gamma subunit of a G 2 breaks down phosphatidylinositol 4,5 bisphosphate forming inositol 1,4,5 trisphosphate (IP 3 ) and diacylglycerol in an enzymatic reaction. Free IP 3 then opens Ca 2+ channels on the endoplasmic reticulum releasing intracellular calcium stores, which leads to the opening of TRPM5 channel s and the subsequent release of neurotransmitters [36] Immunohistochemistry studies have aimed to determine the cell type locations of these pathways. One study determined that gustducin is expressed exclus ively in type II taste cells [38] 2 and TRPM5 are also located in type II taste cells as well, albeit in a larger subset of type II taste cells compared to gustdu cin [37] Consequently, 2 are often used as cell markers for type II taste cells. Double labeling immunohistochemistry with these respective cell markers h ave allowed researchers to identify e xact locations for several

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20 taste receptors. T1R and T2R are both coexpressed with PLC 2 in t ype II taste cells; however, they are not colocalized with each other indicating they are in differ ent subsets of type II cells [39,40] Double immunostaining with GPR120 indicates that it is colocalized with 2 at high percentages [29] Another stu dy used gustudcin to find that CD36 is expressed in type II taste cells as well [8] Researchers continue to use these cell markers to determine the expression pattern of various cell components in taste cells. The signal transduction pathways for salt and sour differ from those utilizing G proteins. The salt taste modality is mostly attributed to the presence of NaCl in foods. It indicates the ingestion of electrolyte rich contents and thus is preferable at low concentrat ions. Thus far, two receptors have been characterized for the recognition of NaCl, EnaC and TRPV1. Both EnaC and TRPV1 are apically located ion channels that allow an influx of Na + into the cell, sufficiently initiating depolarization and neurotransmitter rele ase [41,42] ENaC is referred to as being amiloride sensitive because this sodium channel is blocked in the presence of amiloride [43] Nerve recordings of the chorda tympani reveal that the augmentation of amiloride in the oral cavity does not completely suppress th e salt taste nerve responses [44,45] It is thought that t ide receptor TRPV1 [42] Sour taste is stimulated by acidity and thus the presence of H + ions. This taste modal ity is aversive at high concentrations to prevent the ingestion of spoiled foods The receptors implicated in H + transduction are not well characterized in taste buds ; but receptors from one family are thought to be the primary mediators of sour taste

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21 tran sduction. The receptors PKD1L3 and PKD2L1 of the polycystin 1 (PKD1) like family are both apically expressed toge ther on type III taste cells [26,27] These receptors interact to form a functional channel in sensing HCL. Ablation of taste cells in mice that express PKD2L1 led to reduced responses to sour stimuli as well as an eliminated of nerve respons es in the chorda tympani HCl [26] While it has been shown that these receptors respond to sour stimuli the residual acid responsiveness remaining in mice signal transduction pathway has yet to be fully characterized for sour taste. Neurotrans mitters and Neuromodulators On going research in the field aims to shed light on how taste cells communicate with each other and with sensory nerves to discriminate various signals and relay specific information to the brain for conscious recognition of th e ingested contents. With a labyrinth of taste cells in the taste bud and only a small majority of them containing synapses (both non tradition and traditional) with gustatory nerve fibers, there is an obvious need for cells to communicate with each other to relay information. This phenomenon establishes the utilization of neurotransmitters and neu romodulators in the taste bud for carry ing out such signaling. Interwoven throughout taste buds are innervating gustatory nerve fibers. These fibers project throu gh the basal lamina of the taste bud and surround a majority of the taste cells. Interestingly, it remains that only one of four cell types contains traditional synaptic structures with these nerve fibers. Here enlies the preponder ance of type III taste ce lls [46] Immunostaining has revealed the expression of neural cell adhesion molecules (NCAM), s erotonin and synaptosomal associated protein (SNAP) in this cell type, all of which are molecules related to the presence of synapses in cells. In contrast, type II cells do not contain traditional synapses nor do they contain synaptic molecules;

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22 h owever they do express the majority of taste receptors Interestingly type III cells still produce an action potential in response to taste stimuli even though these stimuli do not directly act upon receptors on type III cells This is probably due to the signal ing that occurs betwee n taste cells [47,48] A possible pathway between these cells is through gap junctions. Gap junctions were identified between type II and type III by dye coupling experiments [49] In theory, upon depolarization type II cells could release secondary messengers to type III cells through these pores. However, there would be a requirement for selectivity of this content to prevent backflow and ensure signal specificity. While this theory provides some explanation for taste cell communication, further and more extensive studies are required to determine the impact of gap junctions in taste cells. Recently the theory for paracrine and autoc rine molecule signaling for cell to cell communication has now been more widely accepted ATP is one of the most studied neurotransmitters in the taste bud. Not only is it a major neurotransmitter for the activation of gustator y nerves [4 6 ], but purinergic receptors are also expressed on adjacent taste cells indicating ATP plays a role in paracrine signa l as well [50] 5 HT is another important neurotransmitter of the taste bud. Antisera against 5 HT determined that it is localized in type III and type IV taste cells [51 53] In taste cells, serotonin is selectively taken up by taste cells through a serotonin transporter and released in a Ca 2+ dependent manner [54] Furthermore, th ere are studies that show cross talk between type II and type III taste cells using ATP and 5 HT. ATP act ivates purinergic receptors on type III cells in response to sweet and bitter stimuli ultimately leading to the secretion of serotonin and the activat ion of afferent nerve fibers [55 57] Additionally,

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23 the serotoni n receptor 5 HT 1A is also expressed on taste cells indicating that it too is involved in cell to cell signaling along with acti vating afferent nerve fibers [58] The list of known neurotransmitters expressed in cel ls of the taste bud is constantly expanding. Other molecules important in taste cell signaling are catecholamines and acetylcholine. RT PCR and immunohistochem i s try reveals that taste receptor cells are an endo g enous source for noradrenaline. Also, a drener gic receptors are expresse d on a subset of taste cells [59] Noradrenaline inhibits K + channels causing an increase in intracellular Ca 2+ [59] These data suggest that catecholamines may play a role in modulating taste sensitivity via paracrine signaling. Finally, ac etylcholine (ACh) is anothe r major taste cell neurotransmitter. One study was able to detect M 1 muscarinic receptor s on a subset of taste cells [60] Receptor activation by ACh leads to an increase in intracellular calcium stores in taste rece ptor cells [60] This indicates ACh possibly has a dual function in t a ste cell communication (i.e. transmission and modulation). Hormone Signaling in Taste buds Deviating from conventional wisdom, a revised view of how information in the taste bud is processed has emerged. Working collectively, taste cells disseminate signaling molecules creating complex pathways to discern abundant and discrete taste qualities. While the list of neurotransmitters in taste buds is extensive, a new frontier is at the f orefront of taste research. The traditional paradigm for taste signaling has now shifted away from cell neuron neurotransmission to cell cell neuromodulation, prior to nerve fiber discharge. A profound impact in this regard was the discovery of neuropeptid e hormones and their receptors in taste buds.

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24 Investigating the function of neuropeptide signaling begins by distinguishing neuromodulators from neurotransmitters. The idea that there are dozens of neurotransmitters and neuromodulators responding to taste modalities might seem unnecessary but the fact that each component plays a contributing role in the ultimate outcome of segregating complex food signals proves their relevance. As with many systems in mammals, modulated pathways include inhibitory and exc itatory communicators. In taste buds, many polypeptide hormones act as neuromodulators by either priming taste cells for signal transduction or inhibiting the cells to preven t them fro m communicating [61] The current paradigm now presumes that polypeptide hormones are while the primary signaling functions are left for the actions of neurotransmitters [62] Multiple experimental approaches have been dev eloped to investigate of h ormonal signaling systems in taste buds. Determining their expression patterns is important for understanding the nature of each respective signaling pathway (e.g. paracrine vs. autocrine signaling) It can also shed light on phys iological actions of these hormones and what tastes qualities they regulate. Using RT PCR, the presence (or absence) of mRNA for the hormones and/or their receptor s can be confirmed. H owever, a deeper investigation is required to determine exact cell locat ions of these hormones. For this, double labeling immunohistochemistry techniques are used to determine their distinct expression patterns Antibodies against these hormones and their receptors can loc ate the exact cell type where they are expressed by col ocalizing these antibodies with known taste cell markers.

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25 A prime example of a hormone that was characterized in taste cells by immunohistochemistry is glucagon like peptide 1 (GLP 1) and its receptor GLP 1R. Expressed in L endocrine cells of the distal in testine, GLP 1 is secreted into intestinal capillaries in response to sweet compounds [63] Immunohistochemistry on L endocrine cells of the intestinal show that GLP 1 is coexpressed with T1R2, T1R3, gustducin, 2 and TRPM5, all of which are common components of the sweet taste transduction pa thway in type II taste cells [63] Indeed, numerous immunostaining studies confirm that GLP 1 is synthesized in type II cells, speci fica lly in a T1R3 subset [64,65] Additionally, the GLP 1 receptor is located on int ragemmal nerve fibers [65,66] D etermining the influence that hormonal signaling has on ta ste perception is rather experimentally challenging Two bottle preferences tests were conventionally used to measure the consumption of taste liquids in the presence (wild type mice) or absence (knockout models) of hormones and/or their receptors. While t his method measures hormonal influences on drinking behavior, the results do not specify the effect s to the oral cavity Experimental confounding variables, such as p ost ingestive effects that occur when stimulus enters the stomach and intestines limit th e conclusions that can be made by two bottle tests. In order to limit these confounders, researchers invented the Davis Rig Gustometer in the 1990s [ 67] Experimentally, the Davis Rig provides brief low quantity access to taste stimuli Reducing the quantity and duration of liquid consumption effectively limits post ingestive behaviors; t herefore, the observed responses are based primarily on signali ng in the oral cavity. When brief access taste tests were performed on GLP 1 knockout mice, a reduced response to sweet stimuli was observed compared to wild type mice.

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26 Interestingly, these knockout mice displayed a modest enhancement of re sponsiveness to sour stimuli [65,66] Based on these data, the study conclude d that T1R3 positive type II taste cells initiate s basolateral secretions of GLP 1 in response to sweet and sour t asting stimuli. GLP 1 then acts upon its recepto r on intragemmal nerve fibers It is possible that this signaling pathway impacts upon the readiness of the nerve fiber and/or relay s ta ste information to the brain [66] As research in fat taste develops, one study has shown that GLP 1 is also playing a role in fat taste modulation [67 ]. Initial indication that GLP 1 is relea sed by taste cells in response to fatty acids was garnered by examining its role in L endocrine cells. It is evident that GLP 1 is secreted by the biological actions of LCF As on the GPR120 receptor in intestinal cells [68 ]. Therefore, since GPR120 is also expressed in taste cells, it was assumed that GLP 1 secreti on would be a consequence of fat taste transduction In fact, Martin et al. 2012 characterized GLP response to LCFA using biochemical, immunohistochemical and behavi oral studies. They concluded that GLP 1 and GPR120 are coexpressed; GLP 1 is released and upon GPR120 activation by LCFAs and this effect is relayed to the GLP 1 receptor on n erves to influence fat taste [68] Both GLP 1 and glucagon are cleaved from the same proglucagon precursor. Glucagon in itself is a hormonal regulator of glucose homeostasis in the liver and pe ripheral tissue, initiating glycogenolysis to break down glycogen into glucose for energy. Similar to GLP 1, glucagon has been found in type II taste cells that express the sweet taste rec 2 [69] The glucagon receptor (GlucR) was also coexpressed in the same cell [69] When glucagon knockout mice were subjected

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27 to brief access taste tests, the results showed a reduced re sponsiveness to sucros e compared to wild type mice [69] A similar reduced response was observed when a Gl uR antagonist was administ ered preceding taste testing [69] Thus, the autocrine signaling of glucagon on cognate receptor is maintain or enhancing responsiveness to sweet stimuli. Modulation of taste sensitivity by leptin was discovered when researchers found enhanced gustatory nerve responses to sweet tastant stimuli in diabetic (db/db) [70] Researchers postulated that this is due to db/db mice having defective leptin receptors. Another study demonstrated a suppressed nerve response to sweet stimuli when leptin was augmented in the oral cavity of lean mice [71] These studies indicated that leptin signaling in the oral cavity is playing a role in the modulation of sweet taste. While leptin itself is not expressed in taste cells, its cognate receptor Ob R on the other hand was discovered by mRNA insitu hybridization [72] Furthermore, it was determined that the presence of leptin reduces the excitability of leptin receptor posit ive taste cells by increasing K + outward currents [72] These di scoveries made opened the door for the possibility that other hormones, produced outside of the taste bud, may be acting in an endocrine manner, entering taste buds and modulating taste responsiveness. Beyond the sweet modality hormonal influence extends to salt, bitter and sour taste perceptions. Insulin is the leading candidate for salt modulation. As previously stated, the sodium channel ENaC is expressed in taste cells and channels the influx of Na + into the cell One of the effects of in sulin signali ng in the gut is the up regulation of ENaC receptors and the permeability [73] If insulin were to be present in taste buds it would be reasonable to assume a similar outcome. Indeed,

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28 patch clamp studies revealed that upon insulin administration Na + influx increased [73] Furthermore and consistent with previous data, this result was depleted in the presence o f amiloride [73] While RT PCR data has revealed that tastes cells express the insulin r eceptor the origin of insulin in taste buds remains unsolved [73] Cho lecystokinin (CCK) was characterized in a specified subset of taste cells using immunostaining techniques. Counting positive immunoreactivity for CCK by confocal microscopy determined there are as many as 14 CCK positi ve taste receptor cells per bud [74] I t is hypothesized that CCK is localized in cells are sensitive to bitter stimuli [74] Furthermore, confirmed by RT PCR, i t was determined that the CCK receptor is completel y coexpressed in CCK positive taste cells [61,74] Acting in an autocrine fashion CCK has the ability to inhibit the outward flow of K + leading to depolarization and intracellular Ca 2+ release. It has not yet been demonstrated what taste is being influenced by the actions of CCK but based on the available data, it is hypothesized that it may be impacting bitter taste functioning. Two more examples of hormonal modulation in taste buds are oxytocin and vasoactive inte stinal protein. An attempt to locate oxytocin by RT PCR demonstrated that it is not expressed in taste cells; rather it is assumed to enter into the taste bud via capillary diffusio n [75] The oxytocin rece ptor on the other hand was found on type I taste cells [75] It was presumed that oxytocin acts as an endocrine hormone on taste buds by influencing type I cells and consequently the structure of the tast e bud however mice deficient in the oxytocin receptor did not show any m orphological alterations [75] There are some studies that suggest it may influence the consumpt ion of sweet and salty foods [76,77] While these studies are suggestive for impacting food consumption,

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29 it is not clear what taste (s) if any, oxytocin modulates. Vasoactive intestinal protein (VIP) is coexpressed with CCK 98% of the time [61] Further localization concluded that a majority of these cells (60%) are immunoreactive for gustducin [61] There is no direct evidence th at VIP affects taste perception. H owever, considering its cellular expression it is a likely candidate for the modulation of bitter perception. Peptide YY and its Signaling in the Oral Cavity Peptide Tyrosine Tyrosine (PYY) is a gastrointestinal peptide hormone. It is synthesized and secreted from L endocrine cells of the distal intestine and colon as well as cells of the pancreatic islet of Lange rhans [78] PYY is part of the n europeptide Y family of hormones that also includes neurope ptide Y (NPY) and pa ncreatic polypeptide (PP). T he gene for PYY is located on chromosome 17q21.1 and following translation a pre pro form of the peptide is made. Following post translational modification, further enzymatic cleavage results in the biologically active PYY 1 36 [79 81] PYY 1 36 contains a poly proline helix, an sheet yielding a folded protein with C and N termin i close together [81] Upon secretion from the cell, PYY 1 36 can potentially be cleaved by an enzyme known as dipeptidyl peptidase IV (DPP IV) yielding another active from of PYY, PYY 3 36 Physiological actions of PYY are mediated by four NPY receptors : Y1R, Y2R Y4R and Y5R [88 ]. U ntruncated residues on the PYY 1 36 polypeptide cause electrostatic interactions with transmembrane helices on the Y1 receptor, altering its confirmation and activating the receptor [82] This renders a higher affinity to Y1R for PYY 1 36 over PYY 3 36 Once cleaved by DPP IV, the C N interactions of the protein are weakened leading to an increase in protein mobility and the formation of PYY 3 36 [80,81] This

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30 mobility allows PYY 3 36 to bind preferentially to the Y2R initiating separate and distinct responses. There are a number of physiological roles for peptide YY including decreasing gut motility, gastric emptying and panc reatic secretions but one of its primar y roles is to induce satiety [83] PYY is secreted from the gu t in response to food intake [78] It is part of an anorexic group of hormones that have been shown to induce s atiety and decrease appetite [83,84] The main anorexic effect takes place when PYY 3 36 travels through circulation cross the blood brain barrier and activates Y2Rs on the arcuate nucleus. PYY 3 36 activates POMC/ a MSH neurons to induce satiety and inhibits NPY/agouti related neurons which decrease appetite [84] These signals are t hen further relayed to the para ventricular nucleus and the periphery where a great er induction of satiety occurs. Recently, it has been shown t hat an alternative p athway for peptide YY exists [84] Acosta et al. 2011 demonstrated by MALDI TOF and RP HPLC that PYY is pres ent in saliva. In concordance with mechanisms for other hormones present in However this possibility has yet to be thoroughly studied. R T PCR of taste tissue located mRNA coding for the PYY protein in the general vicinity of the CVP [84] Interestingly enough, Y1R, Y2R, Y4R and Y5R have all b een found in taste buds as well [85] Immunohistochemistry confirmed the location of PYY and the four Y receptors in taste bud s yet the cell type th at each are expressed in remains unsolved. Further investigations have aimed to determine the behavioral impact of oral PYY signaling [84] Augmenting PYY in saliva, in both acute and long term fashion s caused

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31 anorexic effects similar to its previously known physiological outcomes [84] Consequently, PYY signaling in the oral cavity leads to a premature induction of satiety (to a certain degree) a decrease in food intake and a reduction in body weight overtime. Together, these data provide evidence that o ral PYY signaling is influencing ingestive behavior. One question that remains unanswered is whether oral PYY signaling is impacting taste perception. NPY is part of the same family of proteins as PYY and i s also present in saliva [86] .Unlike PYY, the expression pattern and physiological effects of NPY in taste cells has been extensively studied. Expressed in virtually all of the cells that express CCK (95%), NPY is said to be located in type II taste cells in the specific subset that co expresses T2R [61] Patch clamp studies on isolated taste cells showed that one third of the cells became hyperpolarized in re sponse to NPY administration [61] Hyperpolarization occurred because of enhanced activity for the inward rectif ying K + ion channel (Kir) [61] modulated by NPY, its expression pattern suggests a possible role in bitter taste transduction. Since NPY and the Y receptors are expressed in taste cells, it was hypothesized t hat PYY is present as well. The current study aims to elucidate the role that PYY and its cognate receptors play in mediating taste perception. To accomplish this, I first investigate d the expression patterns for peptide YY and the Y receptors in specific taste cells using double labeling immunofluorescent techniques. Then, using brief access taste tests, I aim ed to determine what taste modalities are being modulated by PYY signaling in the or al cavity. Y1 specific pharmacological approaches were utilized t o determine if the Y1

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32 receptor was mediating one (or more) of the responses seen in PYY / mice. Finally, I used chronic and acute viral vector studies to determine if salivary PYY 3 36 is impacting taste perception and if these affects influence the expres sion pattern of specific NPY receptors.

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33 CHAPTER 2 CHARACTERIZING THE EXPRESSION OF PYY AND THE NPY RECEPTORS IN THE PERIPHERAL GUSTATORY SYSTEM The extent of hormone expression in taste cells has been elucidated in recent decades. The growing list of ne uropeptides expressed in ta ste cells now includes GLP 1 [66] Glucagon [69] Oxytocin [77] CCK [62] NPY [61,87] Ghrelin [64] VIP [61] and Galanin [88] Furthermore, it is apparent that these hormones are expressed in specific taste cell types. For example, GLP 1 is expressed in a subset of type II taste receptor cells that also express taste receptor T1R3 [64] Knowledge regarding the expression pattern of these hormones, as well as that of their cognate receptors, can provide cues as to the model of signaling that each peptide system uses. For example, CCK and its receptor are completely coexpressed in the same cells, indicating CCK signaling may be exhibit autocr ine signaling in taste cells [62] It is important to map these pathways for hormones to determine which cells they are acting upon. T his could shed light on the taste modalities that are being infl uenced by the hormone signaling pathways Expression of the NPY hormone has been extensively stu died in the taste bud. Recently, it has been reported that PYY, another peptide from the NPY family of hormones, i s present in the oral cavity [84] Immunohistochemistry on taste tissue of the CVP determined that PYY is expressed in taste buds. Furthermore, another study uncovered the presence of NPY receptors in taste buds as well [85] Positive immunofluorescence showed Y1R, Y2R, Y4R and Y5R in taste cells and along tongue epithelium [85] While the presences of these proteins were confirmed, it remains undetermined which cell type they are expressed in. In this study, I aim to determine the exact expression pattern of PYY and its receptors using double labeling

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34 immunohis tochemistry. Furthermore, I investigated if PYY signaling in the oral cavity has any influence on the observed expression pattern of the Y2 receptor. Materials and Methods Animal Models and Tissue Collection PYY. NPY KO (129 NPYtm1Rpa/J, Jackson Labs) mice were used to eliminate cross reactivity of our PYY antibody with the structurally similar NPY protein hormone. (Richard Allan Scientific, Kalamazoo, MI) for 8 hours at 4C. The tissue was then dehydrated, paraffin embedded and sectioned at 4 m then stored at room temperature. PYY GFP. Transgenic PYY GFP mice were generated at Duke Universi ty as described by Bohrquez et al. (2010). Eight to twelve week old PYY GFP transgeni c mice were anesthetized and intracardially perfused for 30 minutes with 4% paraformaldehyde prepared in PBS. Following perfusio n, tongue and pancreas tissue were collected and washed once in P BS then fixed in 4% paraformalde h yd e for 4 hours at 4C. After fixation the tissue was cryoprotected at a gradient of sucrose concentrations before being incubated in 30% sucrose overnight at 4C. The following day the tissue was embedded in plastic base molds using O.C.T embedding medium (Tissue Tek, Sakura Finetek Torrance, CA) and frozen in 2 m ethylbutane and d ry ice. Embedded blocks were cryostat (Leica CM3050 S; Leica Microsystems, Nussloch Gmb After the CV was sectioned through, slides were frozen and stored at 80C. For positive control s, colon and pancreas tissue were collected from transgenic mice. For negative controls, wild type C57BL/6J CVP, colon and pancre as tissue was collected in the exact same manner.

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35 NPY R eceptors. Eight to twelve week old C57BL/6J wild type mice were fasted overnight then euthanized and tongue tissue was immediately collected. The Posterior tongue, containing the CVP was excised from the tongue and freshly frozen in O.C.T using 2 methylbutane and dry ic onto Fisherband superfrost plus microscope slides. After the CVP was sectioned, slides were frozen and stored at 80C. All procedures performed on mice were previously approved by the Institutiona l Animal Care and Use Committee (IACUC) at the University of Florida. Immunofluorescence PYY. For PYY immunofluorescence, sections were incubated in 3% H2O2 in methanol to block endogenous peroxidase activity. The sections were then incubated in Trypsin (DIGEST AL L 2, Invitrogen) for antigen retrieval and blocked for 1 hour with 5% natural donkey serum in TNT (0.1 M Tris H Cl, 0.15 M NaCl and 0.05% Tween 20). Following overnight incubation with rabbit anti PYY (Invitrogen, Eugene, Oregon; 1:2,000) at 4C the sectio ns were blocked with Image iT FX Signal Enhancer (Invitrogen). Finally, the signal was detected with donkey anti rabbit Alexa Fluor 488 in TNT (1:1000, Invit rogen), counterstained with DAPI for nuclear staining and visualized using confocal microscopy. St aining on PYY KO tissue as well as omission of the primary antibody resulted in the absence of immunofluorescence. Antibodies and controls used are described in Table 2 3 and Table 2 4. P YY GFP. To determine the cell type of GFP positive cells, double la beling experiments were performed with known ce ll markers for each cell type. GFP immunofluorescence was performed on transgenic PYY GFP tissue. Since the expression o f PYY and consequently GFP is low in taste cells Plus

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36 Fluorescein System (Perkin Elmer, Inc. Waltham, MA) was used to amplify the fluorescent signal. Briefly, slides were allowed to air dry for 30 minutes and post fixed in 10% NBF at 4C for 10 minutes. Antigen retrieval was performed using 0.5% Triton X 100 (ICN Biomedicals, Inc. Aur ora, Ohio) prepared in PBS. Slides were then transferred to 0.02N HCl at ro om temperature for 10 minutes. Sections were blocked using TNB blocking buffer supplied in the TSA kit, blot dried and incubated overnight at 4C with the anti GFP primary antibody (Abcam; 1:500). The following day sections were incubated in Image iT FX signal enhancer for 30 minutes then incubated in Mach 2 HRP for another 30 minutes. Finally, TSA (1:300) was added to the slides for 7 minutes at room temperature and counterstaine d with DAPI for nuclear staining. Every step in this protocol was followed by a few washes in TNT Buffer (0.1M Tris HCl, 0.15M Na Cl, 0.05% Tween 20). Wild type C57BL /6 J CVP and pancreas were used as a negative control. For positive controls, PYY GFP pancre as and colon was collected and the presence of positive immunofluorescence for GFP in expected structures was confirmed. Antibodies and controls used are described in Table 2 3 and Table 2 4. Y1R and Y2R. All tissues were air dried for 30 minutes prior to staining protocols and post fixed in 4% paraformaldehyde for ten minutes. Y1R and Y2R immunolocalization was conducted utilizing the TSA kit. Tissues were blocked in 0.3% H2O2 in TBS for 30 minutes at room temperature to eliminate endogenous peroxidase act ivity, followed by blocking with TNB Blocking Reagent from Perkin Elmer for 60 minutes at room temp to reduce nonspecific antibody binding. Sections were then incubated with the primary antibody (rabbit anti YR) in TNT overnight at 4C. The next day the se condary goat anti rabbit IgG (Fab')2 (HRP) (Abcam; 1:1,000) was added to

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37 the sections for 60 min at room temperature. Staining was detected using fluorescein provided in the TSA kit (1:300 for 7 min at room temp). Negative controls were run concomitantly. All sections were counterstained with DAPI. Antibodies and controls used are described in Table 2 3 and Table 2 4. Double Labeling Immunofluorescence Double labeling techniques were used for the GFP and the NPY 1 receptor immunofluorescent experiments se parately; to determine the type of taste cell t hat each 2 was used as a type II taste cell marker and NCAM was used as a marker for type III taste cells. Following final TSA fluorescein detection of either GFP or the NPY 1 receptor th e sections were blocked and incubated in either anti 2 (Santa Cruz) or anti NCAM (Millipore) overnight at 4C. The following day the slides conjugated Pure Fab Fragment Goat Anti Rabbit) was added for 1 hou r at room temperature. Finally several washes were performed and the tissue was counterstained with DAPI. All final pictures were taken using confocal microscopy. Antibodies and controls used are described in Table 2 3 and Table 2 4 TSA D ouble labeling T echnique The TSA double immunofluorescence technique can be utilized in the instance where two primary antibodies are raised in the same species. In order to prevent the cross reaction between the first primary antibody and the second secondary antibody, t he first primary antibody can be diluted down to a low concentration that is undetectable by a fluorophore conjugated secondary antibody. In this case the antigen can only be detected by the TSA kit and therefore prevents the cross reactivity previously de

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38 fragment in replace of the entire IgG antibody. This controls for the cross reaction that would occur between the second primary antibody and the first secondary antibody that woul d otherwise react unintentionally. This is known as interference I. To control for interference II, after incubation of the Y1 receptor primary antibody the slides were incubated using the second secondary antibody. For this control, no positive Y recepto r staining was apparent indicating the need for TSA amplification in order to detect the Y1 receptor antibody. Interference I is controlled with the use of the experimenta l design there is no occurrence of interference I. By using a second primary antibody in which the antigen is not expressed in tongue tissue but also raised in rabbit I can demonstrate the lack of such cross reactivity. Since Iba is not expressed in in lin gual tissue I used a rabbit an t i Iba (ionized calcium binding adaptor molecule 1) to visualize the possibility of cross reactivity. I replaced the rabbit NCAM and the rabbit PLC 2 antibodies with the rabbit anti Iba1 antibody and the slides were doubled stained as previously described. If interference I was occurring in our staining, the second secondary antibody would be visualized in this control, however, this did not occur. Qu antification of Immunoreactive C ells In order to quantify immunoreactive taste cells impartially, I performed cell stereology procedures similar to previously described systematic approaches [64,76] After tongue extraction, the posterior portion of the tongue was excised. Following fixation and tissue embedding, the CVP was sectioned at 10 m using a cryostat (Leica CM3050 S; Leica Microsystems, Nussloch GmbH, Germany). Tissues were arranged so that every tenth se ction was collected on a single slide, yielding five sections per slide

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39 and taste buds present throughout the entire CVP. A taste bud is approximately 100 m thick so sectioning at 10 m and collecting every tenth section ensure d that no two sections cont ain ed the same taste bud. Then, one slide per mouse was randomly selected using a random number generator for five to six mice per group Staining procedures followed as described above. Confocal microscopy was used to visualize and quantify positive immun oreactivity. Every positive taste cell present on the section was counted. Positive cells were only counted if staining was present in the cytoplasm and specifically around the nucleus to ensure that the contents were present inside the cell. Expression o f Y2R following Acute Augmentation of S alivary PYY 3 36 Concentrations of salivary PYY 3 36 were manipulated to determine its impact on the expression of Y2R in apical locations of the taste bud Eight to twelve week old C57BL/6J wild type mice were fasted overnight (N=24). The next day, mice were divided into fo u r treatment gr oups consisting of n=6 mice per group Group 1 (n=6) was with basal secretions of PYY 3 36 Group 2 (n=6) was fed with normal chow for 30 minutes th and producing native secretions of PYY 3 36 Oral spray (OS) administration of PYY 3 36 (12ug/100BW) was given to group 3 (PYY OS; n=6), followed by a 30 minute incubation, yielding synthetic and acute augmentat ions of PYY 3 36 in the oral cavity The final group (NPY OS; n=6) was administered with NPY (12ug/100BW) oral spra y left fo r 30 minutes, and then euthanized. For control, an additional group (n=6) was given an oral spray of ddH2O to determine if stress fr om the procedure effected expression. Mice were housed at 22 24C in a 12 hours dark/light cycle and had access water ad libitum

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40 These experiments for were approved by Institutional Animal Care and Use Committees (IACUC) at the University of Florida. Fol lowing euthanization, tissues were immediate ly frozen and embedded using 2 m ethylbutane and dry ice. Tissues were sectioned at 10 m and stained with Y2R antibodies. Positive Y2R immunoreactivity and the total number of taste buds were counted using a Leic a Fluorescent Microscope. The percentage of positive immunoreactivity for Y2R was calculated by dividing the number of Y2R positive taste buds by the total number of taste buds per mouse and an average over 5 to 6 mice was calculated Standard T tests were run between groups to determine P values. Scored slides were double checked by a blind observer where randomly selected slides were unidentified, scored, the n rematched with respective treatment groups. Results Confirmation of PYY L ocalization in T aste B u ds The NPY protein contains structural antigens similar to those located on the PYY protein. T his causes minimal cross hybridization of PYY antibodies to the NPY hormone. To control for this I used NPY / mice as our tissue model. The use of these mice fo r such experiments was pr eviously described in Acosta et al. 201 1 [84] Positive immunoreactivity for PYY was observed in taste buds of the collected circumvallate papillae (Figure 2 1A). The manifestation of positive staining seemed more concentrated towards the basolateral portions of the taste bud, but could also be seen in the crux and apex. This manifestation is similar to tha t previously obs erved in Acosta et al. 2011 Figure 2 1B p rovides a loser examination of ibution in a single taste bud. Observed are secret ory granules ( Figure 2 1B; white arrow) contain ing positive immunoreactivity for the PYY protein. These gr anules appear near the

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41 periphery of the cytoplasm by the plasma membrane w h ere it awaits exocytosis and secretion from the cell. These features are consistent with the known distribution of PYY in L endocrine cells [87 ]. Figure 2 1B also shows immunoreac tivity for exogenous PYY located in between and around the taste cells. Mostly taste buds displayed both exo genous and endogenous immunoreactivity as shown in Figure 2 1A B. However occasionally, positive taste buds solely presented endogenous PYY (Figur e 2 1C E), localized within the cells cytoplasm. These positive taste cells displayed large round nuclei, a common anatomical characteristic of type II taste cells (Figure 2 1C). Indeed, 2 (Figure 2 1D E; indicated by whi te arrows) confirming PYY expression in type II cells. Distribution of GFP in T issues of a PYY GFP Transgenic Mouse Model PYY immunofluorescence displayed both endogenous PYY in secret ory granules and exogenous PYY dispersed throughout the taste bud, how ever few taste buds exhibited one or the other. This manifestation made it experimentally difficult to determine cell contours thereby accurately quantifying PYY positive taste cells. Therefore, as a surrogate marker for PYY, I obtained the PYY GFP transg enic mouse model described in Bohrquez et al. 2011. These mice contain a GFP transgene located downstream of the PYY promoter rendering all cells that produce PYY t o simultaneously produce GFP [89] Thus, I continued immunofluores cent experiments using this mouse model to more reliably visualize PYY positive taste cells. Figure 2 2 shows the immunoreactivity of GFP throughout known PYY positive tissues (i.e. pancreas and colon). Visual signal of native fluorescence was enhanced by conventional fluorescent antibody labeling systems. Two major production sites for PYY are cells of the pancreatic islet of Langerhans and L enteroendocrine cells of the

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42 distal intestine and colon. In Fig. 2 2 (A B), I demonstrate that GFP is present in such tissues. Furthermore, the exce r pt in Figure 2 2 B shows a pseudopod like process. This feature of PYY GFP positive L enteroendocrine cells is anatomically similar to what was previously and more thoroughly present ed in Bohrquez et al. 2011 [89,90] Using this tissue, I confirmed the presence of PYY GFP positive immunoreactivity in taste buds of the CVP (Figure 2 1C). For control, I incubated GFP antibodies on wild type C57BL/6J pancreas, colon and CVP tissue, all of which remained negative (Figure 2 2 D F). Quantification of PYY P ositive Taste C ells A closer look at PYY GFP positive taste cells showed GFP immunoreactivity within the cytoplasm of taste cells allowing for the visualization of cell contours and the ability to conduct cell stereology experiments (Fig. 2 3A). Using cell markers for type I I 2) and type III (NCAM) taste cells, I performed a series of double labeling immuno fluorescent experiments to determine and quantify PYY positive cells Fig. 2 3B shows complete co 2, indicated by yellow arrows (right pane l) while a small percentage of positive GFP cells were coexpressed with NCAM (Fig. 2 3C, right panel, white arro ws). Stereological cell counts confirmed our observations. Taste cells that display ed GFP immunoreactivity were co localized with 2 at high percentages (77 % ; S.E.M 1% ) but less frequently (21% ; S.E.M. 3% ) with NCAM (Table 2 1). This data concludes, PYY is primarily expressed in 2 positive type II taste cells while le ss frequently in NCAM positive type III cells. Furthermore, cells co ntaining GFP immunoreactivity displayed large, round nuclei consistent with known anatomical features of type II taste cells.

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43 Y1 Receptors are Primarily Expressed in Type II T as te Receptor C ells The expression of the Y1 receptor in taste tissue has been il lustrated in recent years usi ng immunofluorescent techniques [61,85] These studies utilize a TSA signal enhancement technique to amplify the immunofluorescent signal due to the extremely low expression level of the Y1 receptor in taste receptor cells. Therefore, I used the same technique to characterize Y1R positive taste cells. Figure 2 4 shows immunoreactivity for Y1R s in taste receptor cells of wild type circumvallate papillae tissue. Our findings illustrated Y1R localization inside the cytoplasm of taste cells ( Fig. 2 4A ) making it easy to determine positive from negative cells. I then colocalized this expression with antibodies for known typ e II and type III cell markers. Fig. 2 4B shows a representative taste b ud for Y1R and NCAM colocalization experiments. For the most part, there was an absence of coexpression ; however, a low frequency of co expression was still apparent On the other hand, Fig 2 4C shows coexpression patterns of Y1R 2 in a subset of type II taste cells (right panel; yellow arrows). The white arrow (Fig 2 4C) depicts a small percent of Y1R positive taste cell s that are 2 antibodies. S tereology experiments yielded 77 % (S.E.M 5%) co expression of Y1 2 positive cells. and 11% of Y1R cells were coexpressed with NCAM ( Figure 2 7 ). E ffects of S alivary PYY 3 36 on the Expression of Apical Y2 Receptors in Taste B uds A previously conducted study demonstrated Y2 receptor expression in a pical porti ons of taste buds [85] I have confirmed by TSA immunofluorescent amplification that indeed the Y2 receptor is manifested in such a location (Fig. 2 5). Unlike Y1R, the expression of Y2R manifests itself at anatomical locations consistent with that of the

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44 taste pore (Fig. 2 5D). At this site, I observed positive Y2R immunoreactivity in cellular projections through the taste pore. Because of t he nature of this expression stereological analysis could not be conducted. Due to the presence of Y2R in apical taste cell microvilli, I hypothesized that salivary PYY is int eracting with these apical Y2Rs and affecting its expression. Receptors of the NPY family are known to be internalized and rec ycled upon ligand activation [88 ]. I also nucleus (Fig. 2 5D, white arrow). I hypothesize that this is attributed to the possible immunodetection of inte rnalized receptors or the up regulation of receptor expression due to interactions with peptide YY in saliva ; however, this has yet to be confirmed. It has been shown that PYY 3 36 diffuses into saliv a via leaky capillary action [84] Furthermore, PYY 3 36 displays high affinity for the Y2 receptor. To determine if oral PYY 3 36 is influencing apical Y2R expression I performed Y2R i mmunofluorescence on tissues that contained augmented PYY 3 36 concentrations. To mimic native secretions of PYY 3 36 mice were fasted overnight and then fed for 30 minutes. These mice displayed very little apical immunoreactivity for Y2R ; they did however display an increase in internalized staining ( Fig. 2 6C ; unquantified) Mice that remained fasted (Fig. 2 6D) displayed complete and robust immunoreactivity for Y2R (white arrows). In order to mimic PYY secretions that natively occur after food intake, I augmented PYY 3 36 in the oral cavity by an oral spray method (Fig. 2 6A). Succeeding this augmentation, Y2R immunoreactivity was still apparent albeit much less frequent and with a diminished signal intensity (unquantified) compared to the fasted animal s. Additionally, no significant differences from the fasted group were observed when animals were treated

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45 with NPY OS 12 g/100g BW (data not shown). Furthermore, the control animals (ddH2O OS) revealed identical immunoreactivity to the fasted animals indic ating that stress from the procedure did not affect the results. To quantify the Y2R expression in manipulated mice, the procedure was replicated with n=5 6 mice per group for the fasted, fed, PYY OS and NPY OS groups. The percent of positive taste buds th at displayed apical Y2R expression was calculated ( Figure 2 7 ). Mice that were fed for 30 minutes showed significantly less apical Y2R expression than fasted mice (T test; 44.8% vs. 22.4%; p=0.0028). Similar to fasted BW) displayed significantly more expression of Y2R than fed mice (T test; 46.8% vs. 22.4%; p=0.003). When mice were given an acute administration of PYY 3 36 diminished similar to the fed animals Finally, apica l Y2R expression for the PYY OS group (23.9%) was significantly diminished than the fasted and NPY OS groups (p=0.00057; p=0.00052). Discussion Two ways to define taste cells are by their morphological features and/or the expression of molecules that they contain [15] Taste cells that display round nuclei and 2 are labeled as type II taste cells [15,37] .Taste cells that manifest oblong nuc lei and express NCAM are termed type III taste cells [15,46] 2 and NCAM have been used in numerous immunohistological studies as markers for the ir respective cell types [29,39,46,64,91] It has recently been reported, by RT PCR and confirmed by insitu hybridization and immunofluorescence, that PYY and its cognate receptors (Y1, Y2, Y3 and Y4) are present in taste buds o f the circumvallate papillae [84,85] In this study, I extend previous findings, using double

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46 labeling immunofluorescence with known cell markers to characterize the expression patterns of PYY, Y1R and Y2R in taste cells. Here I demonstrate that antibodies aga inst PYY displayed dispersed immunoreactivity throughout the taste buds (Fig.2 1). PYY positive taste cells that exclusively displayed cytoplasmic PYY reactivity 2 and contained apparent round nuclei. One caveat is that commercia lly available antibodies for PYY display complete cross hybridization between intracellular PYY 1 36 and secreted/enzymatically cleaved PYY 3 36 While it has been shown that DPPIV (the enzyme that cleaves PYY 1 36 ) is absent in taste buds [66] there is still the possibility PYY 3 36 is diffusing into basolateral portions of the taste bu d acting in an endocrine [73] Therefore, it is possible that our PYY antibody is detecting basolateral secretions of PYY 3 36 along with intracellular PYY 1 36 These patterns can be visualized in Acosta. et al. 2011, as well as in Fig. 2 1A. For an accurate quantitative an alysis of PYY positive taste cells, I used circumvallate papillae tissue from a PYY GFP transgenic mouse model. This mouse model provides a better visualization of PYY positive taste cells [89,90] I quantified the frequency of colocalization for PYY 2 expressing type II cells and NCAM expressing type III cells. Stereological experiments determined that 77% S.E.M 1% ex pressed cells are in type II cells and 21%; S.E.M. 3% are in type III cel ls (Table 2 1). In the taste bud, there seems to be a pattern of hormones that are expressed primarily in type II cells but that are also expressed in type III cells. About half of GLP 1 positive taste cells (56%) are positive for gustducin (a cell marke r for a smaller subset

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47 of type II cells) [66] Colocalizaton between the more ge 2 (containing both T1R and T2R subsets) and GLP 1 has not been studied; however GLP 1 could theoretically be expressed in other type II cells as well. Twenty three percent of GLP 1 positive taste cells are coexpressed with t he type III cell marker 5 HT [66] Our data indicates the number of type III pos itive taste cells for PYY is strikingly similar to that of GLP 1 (21 % vs 23% respectively) and definitely within error Indeed, both hormones are expre ssed in L cells of the colon [92] which display many common char acteristics that are app arent in taste cells as well [91] It is now understood that many different hormones are coexpressed together in TCs. For example, virtually of all of the taste cells that express NPY also express CCK and VIP [87] Whether PYY is expressed more or less in a specific subset of type II cells (i.e. T1R or T2R positive) or coexpressed with other hormones has yet to be determined. Further, double labeling immunohistochemistry techniques ne ed to be performed before this comes to fruition expression is the expression pattern for the GPR120 receptor. GPR120 is one of the leading candidates thought to be responsible for the detection of long chain fatty acids i n ta ste cells [9] Matsumura et al. 2009 demonst rated that GPR120 showed 80% co 2, similar to what I f ound for PYY (Table 2 1) [29] Furth ermore, another study showed co expression of GLP 1 with GPR120 and that these cells secrete GLP 1 in res ponse to LCFAs in taste buds [65] The same response can be seen in intestinal L cells of which PYY is also present [68] It is intriguin g to speculate that PYY is coexpressed with GPR120 in taste cells. Indeed, this proposition is currently under investigation in our laboratory.

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48 The current study also confirms the presence of the Y1 and Y2 receptors in taste buds. However, these receptors have markedly different expression patterns. Hurtado et al. 2012 was ab le to detect co localization of Y1R and NCAM in taste cells In this study, I determined that Y1R positive taste cells are coexpressed with NCAM albeit at a rather low percentage (10 % 3% ; Table 2 2). More frequently however, I found that 76% ( 5% ) 2 indicating it is mostly expressed in type II cells. Based on frequency similarities, I suspect that Y1R and PYY might be expressed in the same cell type. However further double labeling experiments need to be condu cted before this is conclusive. Furthermore Hurtado et al. 2011 illustrated localization of Y1R immunoreactivity at apical portions of the taste bud. Using the same antibody and near identical immunofluorescent protocols, I was unable to replicate this data. On the contrary, our staining seemed to display more cytoplasmic localization in taste cells located at the core of the taste bud. Interestingly, I also notice Y1R positive cells a lso located in around type IV cells or basal cells (data not shown). Y1 receptors have been shown in basal cells of the tongue epithelium and are hypothesized to play a role in the maturatio n of tongue epithelial cells Whether or not this holds true for t aste cells remains unknown. I was unable to perform stereological studies for the Y2 receptor due to the limitations of positive immunoreactivity. However, I displayed positive Y2R staining at the apex of the taste bud near the taste pore (Figure 2 6), fu lly corrobo rating data shown in Hurtado et al. 2011. Because of its apparent location, it is probable that these receptors can come in contact with cognate ligands in the oral cavity. It has been shown

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49 that PYY 3 36 is present in saliva and its concentratio n increases after food intake [84] It is also known that PYY 3 36 has a high affinity for the Y2R. Moreover, it is also known th at YRs are internalized upon activation by a ligand [93] I hypothesized that oral PYY 3 36 may be interacting with apical Y2Rs in the taste pore, internalizing them and effectively diminishing their expression, poss ibly even modulating taste transduction pathways. To test if oral PYY 3 36 is influencing apical Y2R expression, I augmented oral PYY 3 36 naturally (by feeding animals) or synthetically (by PYY 3 36 oral spray administration) prior to tissue collection. Inde ed, mice that were fed or given oral administration of PYY 3 36 showed significantly diminished expression of apical Y2Rs compared to fasted animals or control oral spray ( Figure 2 7 ). It is likely that the availability of ligands in the oral cavity are gov erning the expression patterns for apical Y2R (i.e. the absence of PYY 3 36 causes the up regulation of the Y2 receptors). This data also suggests that hormones in the oral cavity may act in an endocrine fashion to influence taste transduction; however, mor e studies on the influence of PYY signaling in taste cells need to be conducted before such a purposed scenario is determined.

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50 Figure 2 1. Positive PYY immunofluorescence in 2 positive type II taste cells. ( a, b, c, e ) Positive immunoreactivity for PYY shown in green of NPY / mice ( a ) The circumvallate papilla sulcus. ( b ) Close up image of a PYY positive taste bud. White arrow indicates positive PYY secretory granules o bserved in cytoplasms Dispersed PYY signaling can be visualized throughout. ( c e ) Exclusive intracellular presence of PYY observed ( d ) The 2 in such taste buds. ( e ) White arrows point to coexpression of PYY and 2. Blue ( a, b, e ) i s DAPI nuclei staining Scale bars: 50 m ( a ); 10 m ( b c e ). (c) (d) (e) PYY PLC 2 Merg e

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51 Figure 2 2. GFP immunoreactivity throughout PYY GFP transgenic murine t issue Positive immunoreactivity for GFP (green) was observed in pancreatic cells ( a ), colon ( b ) and sulcus of the CVP ( c ) in transgenic PYY GFP mice. Exce r pt in ( b ) displays a GFP positive pseudopod like L endocrine cell. Wild type C57BL/6J tissue was negative when incubated in the GFP antibody ( d f ). Blue is DAPI nuclear staining. Scale Bars: 50 um ( c, f ); 100 m ( a, d ); 200 m ( b, e ) Wild Type Pancreas (d) Wild Type CVP Trench (f) (c) PYY GFP Trench (a) PYY GFP Pancreas (b) PYY GFP Colon (e) Wild Type Colon

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52 Figure 2 3. GFP double labeling immunofluor scence in CVP taste buds of PYY GFP transgenic mice. ( Panel a ) PYY GFP immunoreactivity displayed exclusive cytoplasmic localization within taste cells. ( Panel b ) Co loc alization of PYY GFP positive cells with 2 (right; yellow arrows). ( Panel c ) Expression of PYY GFP and NCAM. Low frequency colocalization (right; yellow arrow) and high frequency uncolocalized GFP PYY cells (right; white arrows). Blue is DAPI nuclear staining. Scale bars: 10 m ( Panel a c ).

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53 Figure 2 4. Co expression of Y1 receptor with 2 positive type II taste c ells ( Panel a c ) Circum vallate papillae taste buds of C57BL /6 J mice. ( Pane l a ) Positive Y1R immunoreactivity was determined by TSA ampli fication systems (green). Localization of Y1R manifested in taste cells cytoplasm. ( Panel b ) Infrequent immunoreactivity for Y1R and NCAM. ( Panel c ) Co expression of Y1R with 2 (right panel; yellow arrow) and white arrow indicates Y1R positive cell wit h no coexpression Blue is DAPI nuclear staining. Scale bars: 10 m (Panel a, Panel b c ) (a) Y1R NCAM Merge DAPI Y1R Merge Y1R PLC 2 Merge (b) (c)

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54 Figure 2 5. Localization of Y2 r ecept or expression in apical taste pores of murine taste b uds. ( a c ) Circumval late papillae sulcus tissue of C57BL /6 J mice con taining several taste buds. ( b d ) Positive Y2R immunoreactivity visualized at anatomical locations consistent with taste pore sites. ( d ) Close up image of an exemplified taste bud displaying apical Y2 receptor immunoreactivity. White arrow ( d ) denotes slig htly positive presence of intracellular Y2R. Scale bars: 50 m (a c); 30 m ( d ). (a) (b) (c) DAPI Y2R Merge ( d) Close up

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55 Figure 2 6. Expression of apical Y2 receptor in the t ast e bud based on metabolic s tate. ( a d ) Circumvallat e papillae sulcus tissue of C57BL /6 J mice. Y2 receptor positive immunoreactivity shown in green. ( a) Oral administration of PYY 3 36 (12 g/100g body weight). ( b ) Control mice were administered with ddH2O. ( c ) Mice were fed, mimicking endogenous secretions o f PYY 3 36 following food intake. ( d ) Fasted mice displayed greatest Y2R immunoreactivity at tas te pore locations White arrows designate positive apical Y2R staining. Scale Bars: ( a d ) 50um PYY OS H2O OS Fasted Fed (a) (b) (c) (d)

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56 Figure 2 7. The effects of oral administration of PYY 3 36 on apical expression of Y2R in taste buds. Mice were fasted mice (n=6) or fed (n=5; 30 min) prior to tissue collection. For fasted mice, 44.8% ( SD 9.8) of taste buds displayed positive immunoreactivity for Y2R. For fed mice (n= 5) 22.4% ( SD8.6 ) of taste buds were Y2R positive. Two groups were given an acute administration of PYY OS or NPY OS displayed 23.9% ( SD 5.1 ) and 46.7% ( SD 7.1 ) immunoreactivity in their taste buds r espectively. **P < 0.01, ***P<0.001

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57 Table 2 1. Percentages of GFP positive taste cells colocalized with cell markers. Percentages were determined by dividing the number of colocalized cells 2 or NCAM) by the total number of PYY GFP positive c ells yielding the percent of PYY cells that are expressed in respe ctive cell types. On average, 77 % ( S.E M. 1% ) of PYY GFP positive taste cells w ere also 2; 21% ( S.E.M. 3% ) of PYY GFP positive taste cells were positive for NCAM. PY Y GFP 2(Type II cells) Co expressed CO/Total PYY GFP positive cells (%) Ms 1 31 27 229 88% Ms 2 43 40 287 87% Ms 3 48 28 218 82% Ms 4 68 12 331 82% Ms 5 59 8 353 86% Total 249 115 1418 85% Average 83 38.33 283.6 77% S.E.M 6.40 5.81 26.79 1% PYY GFP NCAM (Type III cells) Co expressed CO/Total Y1R positive cells (%) Ms 1 226 152 45 12% Ms 2 199 180 43 18% Ms 3 213 191 56 20% Ms 4 160 153 63 28% Ms 5 235 182 69 22% Total 1033 858 276 21% Average 206.6 17 1.6 55.2 21% S.E.M 13.14 8.02 5.02 3% NCAM 2 PYY GFP (%) 21 3 77 1

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58 Table 2 2. Percent ages of Y1R positive taste cells colocalized with cell markers. Percentages were determined by dividing the number of colocalized cells 2 or NCAM) by the total number of PYY GFP posi tive cells yielding the percent of PYY cells that are expressed in respe ctive cell types. On average, 77 % ( S.E.M 5%) of PYY GFP positive taste cells were also positive for P 2 and 10 % ( S.E.M. 3%) of PYY GFP positive taste cells were positive for NCA M. Y1R 2 (Type II cells) Co expressed CO/Total Y1R positive cells (%) Ms 1 29 47 154 84% Ms 2 21 25 198 90% Ms 3 62 23 188 75% Ms 4 62 31 207 77% Ms 5 120 13 186 61% Total 232 139 933 80% Average 58.8 27.8 186.6 77% S.E.M 1 7.44 5.61 8.98 5% Y1R NCAM Co expressed CO/ Total Y1R positive Cells (%) Ms 1 260 140 39 13% Ms 2 165 104 36 18% Ms 3 208 157 18 7% Ms 4 165 149 13 7% Ms 5 147 140 6 4% Total 945 690 114 11% Average 189 138 22.4 10% S.E.M 20 9 6 3% NCAM PLC 2 Y1R (%) 10 3 76 5

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59 Table 2 3 Antibodies used in Immunohistochemistry experiments Primary antibody Species Source Dilution Detection Source Dilution PYY Rabbit Abcam 1:2,000 Alexa Fluor 488 Invitrogen 1:1,000 GFP Rabbit Abcam 1:500 M ach 2 Rabbit HRP Polymer + TSA Abcam 1:1,000 Neuropeptide Y1 Receptor Rabbit Immunostar 1:300 Mach 2 Rabbit HRP Polymer + TSA Biocare Medical and Perkin Elmer 1:300 (using TSA kit detectio n) Neuropeptide Y2 Receptor Rabbit Neuromics 1:2,000 Mach 2 Rabbit HRP Polymer + TSA Biocare Medical and Perkin Elmer 1:300 (using TSA kit detectio n) PLC 2 Rabbit Santa Cruz 1:200 Cy TM 3 conjugated Pure Fab Fragment Goat Jackson ImmunoResearch 1:1,000 NCAM Rabbit Millipore 1:1,000 Cy TM 3 conjugated Pure Fab Fragment Goat Anti Rabbit Jackson ImmunoResearch 1:1,000 Table 2 4. Control experiments performe d for Antibodies Antibody Controls PYY Staining negative with the omission of the primary antibody. The staining of PYY KO tissue yielded positive results due to cross reactivity with the NPY protein. NPY KO tissue was used in this study to control for th is. This antibody and method was described in Acosta et al, 2011 PLoS ONE 6(10): e26137. GFP Staining negative with the omission of the primary antibody. Wild type c57bl/6 CVP and pancreas tissue was used as negative controls, both yielding negative stain ing. NPY receptors Staining negative with the omission of the primary antibodies. Staining specific to hippocampal regions of the brain in positive controls. Thi s antibody was used in Acosta et al, 2011 PLoS ONE 6(10): e26137. PLC 2 Staining negative wi th the omission of the primary antibody NCAM Staining negative with the omission of the primary antibody

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60 CHAPTER 3 TASTE MODULATION BY PYY SIGNALING The growing list of hormones and their receptors that are expressed in taste cells begs the question o f whether they have any impact on taste functioning. Their expression patterns suggest possible pathways putative for cell to cell communication abilities in the taste bud However, while their localization may insinuate potential i nfluences on taste perce ption, that information alone does not prov ide strong enough evidence for any functional impact. As the view on how the taste bud functions continues to evolve [61] it is now believed that polypeptide h ormones may play a role in the modulation of taste responses Addit ional studies of hormone signaling will be required for a complete understanding of the influence of each hormone on taste perception. The little data that are available suggest that some hormone signaling systems affect taste bud function by mediating the electrical excitability of taste cells. Their signaling can effectively excite or mitigate intrinsic properties of taste cells and impact their cellular outputs. In this way, it is currently believed that hormone modulators play key roles in taste percept ion. It has been shown that hormones can act in an autocrine, paracrine and endocrine manner in the taste bud to impact taste functioning. For example, leptin is considered an endocrine taste hormone because it is not expressed in taste buds yet it activa tes leptin receptors expressed in a subset of taste cells [72] Patch clamp studies showed that peripheral administration of leptin on taste cells caused activation of outward K + currents re sulting in hyperpolarization [72] Leptin is just one of a few hormones that enter the taste bud through circulation and impacts taste function. Hormones can also act in an autocrine manner, imp acting the excitability of its own cell.

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61 For example, s ignaling by CCK on its receptor CCKA (expressed in the same taste cell) results in the inhibition of outward K + affectively influencing the polarization of its own cell [61] Finally, hormones such as GLP 1 and NPY can act through their receptors on neighboring taste cells or innervating nerve fibers, to impact taste transdu ction by paracrine signaling [61,66] In the past, determining the taste modalities that are influence d by hormone signaling was challenging to study. Studies util ized two bottle taste tests using gene knockout animal models to determine if taste behavior was being impacted by the interruption of a given hormonal signaling system However, these tests are somewhat limited in nature. P ost ingestive effects confound measure behavior exclusively based on signaling within the oral cavity. The Davis Rig gustometer was developed to limit the influence of the se variabl es on t aste related behavior [67] Researchers now routinely use this device to determine the impact of oral hormone signaling on taste guided behaviors F or example, a recent study detailed the use of a brief access taste test using the Davis Rig gustometer to determine that GLP 1 is modulating sweet and s our taste perception in mice [66] Another study demonstrated the impact of glucagon signaling on sweet taste perc eption using similar methods [94,95] In the current study, I aimed to shed light on the taste(s), if any, which are being impacted by PYY signaling. I performed brief access taste tests, using the Davis Rig gustometer on PYY / mice Furthermore, I used a pharmacological approach t o determine what receptors are mediating the responses. Lastly, I utilize novel gene therapy techniques to study exact impacts of salivary PYY 3 36 on taste perception.

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62 Materials and Methods Animals For behavioral studies te n to twelve week old wild type C 57BL /6 J mice (n= 8 10) served as a control group for PYY / mice (n=8 10). Upon arrival, mice were separated and housed individually in standard cages with bedding. The colony room was temperature consistent and light controlled for a 12 hour on, 12 hour o ff light cycle. Mice were acclimated to their environment for 7 days prior to testing and were fed ad libitum for the duration of this period. Mice were then put on water restriction (23.5 hours) intermittently during the course of experimental testing. At any time, if mice dropped below 85% of their body weight, they received 1 ml of supplemental water 2 hours after a testing session. All procedures were previously approved by UF IACUC. Taste Stimuli Taste stimuli was prepared in purified H 2 0 (Elix 10; Mil lipore, Billerica, MA). All taste chemicals were reagent grade and presented to the mice at room temperat ure. Presentations encompassed a variety of concentrations for each tastant. Intralipid Emulsion (fat stimuli) was diluted at 1.25%, 2.5%, 5%, 10% and 20% (Baxter Healthcare, Deerfield, IL). Intralipid is comprised of linoleic (44 62%), oleic (19 30%), palmitic (7 14%), linolenic (4 11%) and stearic (1.4 5.5%) Corn oil (fat stimuli) was diluted at 2.5%, 5%, 10%, 30% and 40% (Mazola, Best Foods, Englewoo d Cliffs, NJ). Mazola corn oil is comprised mostly of linoleic acid (47%), Oleic acid (36%), Steric acid, (11%), Palmitic acid (12%)Sucrose (sweet) was diluted at 25mM, 50mM, 100mM, 200mM and 400mM (Fisher Scientific, Atlanta, GA, USA). NaCl (salt) was dil uted at 30mM, 100mM, 200mM, 300mM, 600mM and 1M (Sigma Aldrich, St. Louis, MO, USA). Denatonium Benzoate (bitter) was diluted at 0.01mM, 0.03mM, 0.1mM, 0.5mM and

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63 1.5mM (Sigma Aldrich, St. Louis, MO, USA). Quinine Hydrocholide (bitter) was diluted at 0.01mM 0.03mM, 0.1mM, 0.5mM and 1.5mM (Sigma Aldrich, St. Louis, MO, USA). Citric acid (sour) was diluted at 0.3mM, 3mM, 1 0mM 30mM and 100mM (Sigma Aldric h, St. Louis, MO, USA). Tastant concentrations are consistent with previously studied ranges [66,69] A separate set of mice were used for pharmacological studies. Briefly, Y1 receptor antagonist BIBO 3304 trifluoroacetate (Tocris Bioscience, Minneapolis, MN, USA) was prepared in DMSO (Sigm a Al d rich). The receptor antag onist cocktail was and administered to each mouse. Chronic Salivary Augmentation of PYY 3 36 Cassettes encoding pre pro PYY or GFP were pseudo typed into the recombinan t adeno associated virus serotype 5 capsid (Fi g. 3 1; rAAV5 PYY; rAAV5 GFP). Following vector production and purification, sufficient titers for vector delivery were determined (10 12 viral particles/mL). Vectors were injected into PYY / mice solely recons tituting salivary PYY 3 36 [64,84] Chronic salivary augmentation was achieved by submandibular salivary gland injections. Ductal cavities of the gland were cannulated by polyethylene tubing (PE10; BD intramedic, Ont ario Canada). Virus was diluted down to 1x 10 10 vp/mL PYY (n=10) or rAAV5 GFP (n=10) was injected into both glands of the mice. Mice were housed ad libit um for 1 month prior to Davis R ig testing. GFP immunostaining in salivary gland cel ls confirmed effective viral infection (data not shown). These procedures were previously described in Acosta et al. 2011 and Hurtado et al. 2012 [84]

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64 Brief access Taste T ests Brief access taste tests took place in a Davis Rig Gustometer (Davis MS 160l DiLog Instruments, Tallahassee, FL, USA; Smith, 2001). Sipper tubes are prepared with aforementioned solutions and arranged into a ccess slots in the Davis Rig. Water deprived mice are restricted access to the tubes for 5 second presentations w h ere they are allowed to lick tastants at their will. Two protocols, one for naturally preferred substances and the other for naturally avoided substances, were used for brief access taste testing. Mice that were tested with preferred stimuli were restricted of food and water for 23.5 hours prior to testing and given 1g of food and 2ml of water to maintain healthy status Prior to food restrictio n they were given 23.5 hours of food and water ad libitum to recover. For naturally avoided stimuli mice were restricted water for 23.5 hours on all testing days to encourage ingestion. Five second interpresentation lick intervals of H 2 O followed each pre sentation to minimize crossing over effects. Each test session lasted 25 minutes. Statistical Analysis Statistical methods presented quantify the diff erences in stimuli responsive ness for each genotype. The average number of licks per trials was divided by average water licks per trial generating a tastant/water lick ratio. Each ratio was analyzed by group/genotype x concentration analyses of variance (ANOVA). The difference between lick ratio s revealed by concentration by genotype interaction were assessed using t tests. The statistical rejected criterion was set p 0.05. Mice were to the mean data for each group/genotype using a logistics function described in Equation 3 1:

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65 (3 1) x = log 10 concentration at the inflection point, c= log 10 concentration at the inflection point, b =slope, a = asymptotic lick ratio, d = minimum asymptote of lick ratio. Results PYY Signaling Modulates Fat and B itter Taste Responsiveness in M ice. To determine if PYY signaling affects taste responsiveness I performed brief access taste tests using the Davis Rig gustometer No apparent morphological abnormalities were observed in knockout compared to wild type tas te buds. During brief access taste testing, mice received a range of concentrations for sweet, bitter, sour, salty and fat stimuli. Tastant to water lick ratios were compared for wild type vs PYY / mice as a function of concentration. PYY / mice were le s s responsive intralipid emulsion (p=0.0009, interaction) and corn oil (p=0.01) and the bitter stimuli denatonium benzoate (p=0.04) and Quinine Hydrochloride (p=0.01) (Figure 3 1). No significant differences were observed for sucrose, NaCl or c itric acid. These data indicate that the ability to respond to fat and bitter tasting stimuli Thus, PYY signaling seems to be influencing fat and bitter taste modalities. Impac ts of Y1 Receptor on T aste R esponsiveness To determine if the Y1 receptor is mediating one (or both) of the observed responses, I performed a pharmacological study using the Y1 receptor antagonist BIBO 3304. The Y1 receptor antagonist was integrated into taste solutions and administered to mice in a brief access taste test. Mice who received the receptor antagonist showed a significant reduction in their proclivity to respond to bitter stimuli (p=0.007) compared

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66 to vehicle treated mice (Figure 3 2). Furthe rmore, mice actually showed an increase in responsiveness towards fat emulsion (p=0.003). No significant differences were observed for the stimuli sucrose, NaCl or citric acid. This data suggests that the Y1 receptor is mediating responsiveness to bitter t asting stimuli either through the actions of PYY or NPY signaling. Also, this data indicates that blocking Y1 receptor signaling causes hypersensitivity to fat stimuli. The E ffects of Salivary PYY 3 36 on F at Taste P erception Recombinant adeno associated viral vectors have recently been used as a method to chronically overexpress PYY in saliva [84] PYY / mice were injected with 5 0 10 vp/mL, directly in to the submandibular salivary glands. PYY secretions from targeted salivary gland cells exclusively secrete PYY into saliva without increasing the concentration of PYY in circulati on [84] Therefore, by using global PYY knockouts and administering rAAV PYY to the salivary glands; this method ensures that PYY signaling occurs solely in the oral cav ity. Salivary glands from vector injected animals displayed no gross distortions in sal ivary gland anatomy [84] Furthermore, no t only is PYY signaling reconstituted in the oral cavity, but there is a 2 to 5 fold increase of PYY 3 36 circulating in saliv a compared to wild type mice rAAV GFP mice were used as a control group to confirm the effective gene delivery. Mice treated with PYY vectors showed a significant increa se in responsiveness towards Intralipid emulsion (p=0.005; interaction) compared to the control rAAV GFP treated mice (Figure 3 2), thereby rescuing the fat taste response in PYY / animals. On the other hand, vector treated mice showed no significant difference in the ability to respond to denatonium benzoate. This data strongly suggests that salivary PYY 3 36

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67 Discussion Here I report oral PYY signaling modulates the responsiveness of mice to two known taste modalities: fat and bitter. I utilized PYY / mice that have a global knockout of PYY, effectively abolishing any potential PYY signaling that occurs locally in the oral cav ity or peripherally in other tissues. When these mice were subjected to a brief access taste test, PYY / mice exhibited a significant reduction in taste responsiveness to fat (Intralipid emulsion, corn oil) and bitter (denatonium benzoate, quinine hydroc h loride) stimuli (Figure 3 1). Intralipid emulsion is a concoction of essential fatty acids aimed to mock dietary fats. It consists of a variety of fats including linoleic acid (C 18 ), oleic acid (C 18 ), palmitic acid (C 16 ), linolenic acid (C 18 ) and steric ac id (C 18 ). These comprise a list of a few of the most common fatty acids found in most dietary fats as determined by the Standard American Diet [96] By definition these fats are all considered long chain fatty acids (C 13 C 21 ). PYY / mice showed a reduced proclivity to respond to these long chain fatty acids (LCF As) compared to wild type (normal PYY signaling) mice (Figure 3 1). Further strengthening our conclusions, these mice displayed a reduced response for corn oil which contains a similar composition to that of I ntralipid (47% linoleic and 36% oleic acid). Wh ile this data suggests that PYY plays a role in modulating LCFA sensitivity, I have yet to determine if there is a single fatty acid with in these mixtures contributing to these behaviors or if there are middle and/or short chain fatty acids contributing to this effect In addition, PYY / mice also showed a reduced proclivity to respond to bitter tasting stimuli (Figure 3 1). It is interesting to note that T2Rs, commonly referred to as the bitter taste receptors, are expressed in L cells of the colon that contain both PYY

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68 and GLP 1 [97] Calcium imaging studies have shown that enteroendocrine cell lines increase intracellular calcium and release hormones upon bitter tastant stimulation [98,99] Assuming that enteroendocrine cells and taste cells are alike, it is possible given our data, that PYY is being secreted from T2R positive taste cells in response to bitter intake and is modulating bitter taste responses. In order to de termine what tastes are being modulated by the Y1 receptor I performe d a brief access taste test on C57BL /6 J mice using a Y1 receptor antagonist intermixed with the tastants (Figure 3 2). Y1 receptor antagonist BIBO 3304 was developed to exhibit 100 fold affinity for the Y1 receptor without being pharmacologically act ive for other NPY receptors [99] Mice that received the Y1 a ntagonist displayed a reduced response to denatonium benzoate indicating, at least in part, that the Y1 rece ptor modulates bitter taste perception I attribute this to lack of the Y1 receptor availability for PYY signaling. Adding to the systems complexity, NPY is also present in taste cells and acts on the Y1 receptor [87] Previous studies have demonstrated that the activation of Y1 recept ors by NPY in taste cells causes an increase in the inward rectifying potassium current (Kir) effectively stabilizing the resting potential of the cell and a t tenuating its excitability [87] Furthermore, no increases in intracellular calcium stores were observed in these cells either [87] It is common a characteristic of the NPY family receptors that NPY and PYY 1 36 (uncleaved) exerts the same effects on the Y1 receptor in many physiological systems [100 102] Due to the abse nce of DPPIV [66 ], PYY that is expressed in taste buds remains uncleaved. In this study, I extend previous data and conclude that the attenuation in excitability in Y1R positive taste cells [87] by PY Y 1

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69 36 /NPY signaling leads to our observed diminished responsiveness towards bitter stimuli. It would be interesting to perform further co labeling studies of the Y1 receptor and T2R, to determine if in fact PYY 1 36 /NPY signaling is commiserating with bi tter sensitive cells. Furthermore, mice given the Y1 receptor antagonist showed a significant increase in responsiveness to intralipid fat emulsion (Figure 3 2). Inhibiting the Y1 receptor and therefore increasing the excitability in lipid sensing cells co uld lead to the hypersensitivity responses I observed. This model assumes Y1R and lipid receptors are coexpressed on the same cell which has not yet been experimentally determined. Another possibility is that this response is mediated by another candidate of Y receptors (i.e. Y2R). Indeed, opposing effects of Y1 vs Y2 activation have been tho roughly studied in other mammalian systems [85,101] Therefore, blocking the effects of the Y1 receptor could lead to increase d ac tivation of the Y2 receptor [101] To determine the impact of salivary PYY acting as an endocrine hormone on taste responsiveness I performed a viral vect or experiment. R ecombinant associated viral vector s have been used recently as a novel mechanism to reconst itute PYY in the oral cavity [84] I demonstrate that the augmentation of PYY sign aling in the oral cavity leads to a rescue in responsiveness to wards fat but not bitter tasting stimuli (Figure 3 3). Therefore, I hypothesize that salivary PYY is modulating fat taste responsiveness. A gene transfer of PYY into cells of the submandibular salivary gland of PYY / mic e produces a 2 to 5 fold increase in PYY concentrations in saliva [84] This production heightens P YY signaling in the oral cavity and creates more robust effectual responses. In fact, previous studies showed an increase in PYYs anorexic effect when

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70 infected with rAAV PYY in the s alivary glands of KO animals [84] It is important to note that the enzyme DPPIV is present in saliva and therefore secretions of PYY 1 36 are quickly converted to PYY 3 36 [84] It is also known that PYY 3 36 displays the highest affini ty for the Y2 receptor [81,103] In this communication, and in conjunction wi th previously published data [85] Y2 receptor expression is localized on apical portions of the taste bud. It is reasonable to assume that apically projected Y2 receptors can interact with salivary PYY 3 36 modulating the taste responsiveness for fat stimuli. Furthermore, it has been demonstrated by calcium imaging that salivary PYY 3 36 causes an increase in intracellular calcium on lingual epithelial cells [85] Assuming that a similar signal transduction cascade occurs in taste cells, PYY 3 36 has the potential to increasing intracellular calcium stores on Y2R positive taste cells and, as a result, influence fat taste perception. Overall, it is interesting to speculate what these findings suggest to the greater scope of obesity. In humans, fat taste preferences and fat taste perception can contribute to the development of obesity [104] Human subjects, who are obese, tend to have a greater sensory preference for fatty foods than their lean counterparts [28] Stewart et al. 2010 further demonstrated that humans with higher BMIs were less sensitive to LCFAs and consumed more fat than humans who were more s ensitive to LCFAs [105] Extending these findings to other mammalian species, mice that are less sensitive to orally administered fats, consumed more fat in their diets and develop obesity [106] In this study, I demonstrate that mice lacking PYY signaling in the oral cavity, do not fully respond to LCFAs. The decrease in fat preference could cause subjects with lo wer levels of salivary PYY 3 36 to consume more fatty foods. The

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71 converse relationship has already been demonstrated by Acosta et.al. 2011, that by increasing PYY signaling in the oral cavity, they observed a decrease in HF diet intake and a decrease in bod yweight. Clinical trials are currently underway to determine if obese humans have lower levels of salivary PYY 3 36 than lean subjects. The current study provides a possible mechanism by which this behavioral observation is occurring. The most recent evide nce points to apically expressed taste receptors as the contributing mechanism for differences in fat intake [106] Indeed, varying levels of taste receptor expression influences taste preference and results in d ifferent patterns of fat intake For example, the CD36 receptor, responsible for detecting dietary LCFAs in taste buds, has been shown to be down regulated in circumvallate papillae taste buds of diet induced obese rats [106] Adding to these data my study demonstrates a decrease in apical Y2 receptors in response to salivary PYY 3 36 as a result of food intake. If Y2 receptors are in fact down regulated with chronic presentations of salivary PYY 3 36 overtime, then our data suggests that these mice would be less sensitive to fatty foods and chronically consume more fat.

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72 Figure 3 1. Diminished responsiveness of PYY / mice to fat and bitter tasting stimuli. Brief access taste test on PYY / ( o pen circles; n=8) vs WT mice (filled ci rcles; n=8). PYY / mice globally abolishes PYY signaling, native to WT mice Response is expressed as a tastant to water lick ratio as a function of stimulus concentration. Each point is expressed as means of SE. Das hed lines at 1.0 are ratio for water. Concentrations are presented by mM or percent ranges. PYY / mice showed a significant reduction in sensitivity to denatonium benzoate (p=0.04) QHCl (p= 0.01), as well as corn oil (p= 0.01 ) and I ntralipid emulsion s (p=0. 0009 ) at presented concentrations. N o significant differences were observed for sucrose, NaCl or citric acid

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73 Figure 3 2. Y1 receptor mediated responses to taste stimuli. Y receptor antagonist BIBO 3304 mixed into each stimulus (1mM; open circles) w as administered to C57BL /6 J mice in brief access taste tests. Each point is expressed as means of SE. Dashed lines at 1.0 are ratio for water. Concentrations are presented by mM or percent ranges. Compared to vehicle control (DMSO; filled circles), antagon ist mice showed reduced responsiveness to denatonium benzoate (p=0.015), and an increase in responsiveness to I ntralipid emulsion (p=0.003) Excerpt in the I ntralipid graph displays the validation for tastelessness of BIBO 3304.

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74 Figure 3 3. Saliva ry PYY 3 36 modulates fat taste perception Submandibular salivary glands of PYY / mice were injected with rAAV5 PYY (filled circles; n=8) or control rAAV5 GFP (filled circles; n=8) and subjected to a brief access taste test. Each point is expressed as mea ns of SE. Dashed lines at 1.0 are ratio for water. Concentrations are presented by mM or percent ranges. Mice with oral PYY 3 36 augmentation displayed increased responsiveness to Intralipid emulsion (p=0.00002 ) but not to denatonium benzoate compared to co ntrol

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75 CHAPTER 4 CONCLUSIONS AND GENERAL DISCUSSION The NPY family of proteins consists of NPY, PYY, PP and their cognate receptors; Y1R, Y2R, Y4R and Y5R expre ssion of NPY in salivary glands and evidence s uggesting i ts presence in saliva was unveiled soon thereafter [86] In recent years, patterns of expression for these proteins have been revealed in novel physiological domains. A myriad of h ormones, including PYY, were discovered in saliva as well Following the ir discovery, investigations revealed that Y receptors were also expressed in cells of the oral cavity. RT PCR and immunofluorescence unveiled distinct pa tterns of NPY receptors amasse d in stratified layers of lingual epithelium [85 ]. The discovery of these proteins in th e oral cavity led researchers to investigate t he physiological impacts of these signaling system s The metabolic consequence of PYY 3 36 signaling on Y2 receptors in the arcuate nucleus is the suppression of appetite a nd promotion of weight loss [101] In the spirit of a physiological commonality, it is now known that PYY exerts similar satiation effect through an alternativ e pathway in t he oral cavity [84] Research on gast rointestinal hormones extended from the expression of these peptides in oral tis sue, in gen eral to their expression in th e peripheral gustatory system, in particular. A recent breakthrough for this system was the discovery of NPY in peripheral gustatory tissue s In 2005, the expression patterns for NPY we re examined in rat taste cells [87] Its physiological actions influence taste cell excitability, functionally impacting signal transduction systems [93 ]. The divulgence of other NPY family proteins in peripheral gustatory tis su e followed soon thereafter [84,85] RT PCR and

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76 immunofluorescence evinced the expression of PYY and four of the Y receptors in taste buds [84, 85 ]. While sufficiently ascertained, this story remained incomplete. Th e expression patterns of PYY and the Y receptors in taste buds were unknown. The physiological impacts of P YY signaling in taste cells had not been investigated and its influences on taste perception remained undetermined I init iate d investigations to enlighten the final pieces of this puzzle. Based on evidence presented in Chapter 2 and Chapter 3 of this thesis, I extend the known origins of PYY and the Y receptors in taste buds by characterizing their expression patterns and pi npointing their locatio ns to specific taste cell types Using contemporary methods, I appraised the taste modalities being impacted by PYY signaling. Finally, I have developed a contingent model for PYY signaling in the peripheral gustatory system. Charact erizing t he Expression of PYY and the NPY Receptors in the Peripheral Gustatory S ystem in taste buds. By PYY immunofluorescence, I confirmed the presence of PYY in CVP tissue of mice. PYY immunoreactivity exhibited both endogenous PYY, located in secretory granules, and exogenous PYY, located around plasma membranes. The taste cells that solely displayed endogenous PYY staining were co 2. To quantify this da ta, I performed stereology experiments using double labeling immunofluorescence on transgenic PYY GFP tissue. A known cell marker for type II 2 [43] I found that taste cells that expressed PYY GFP also 2 at a frequency of 85%. NCAM is a cell mar ker for type III taste cells [51] I determined that PYY GFP positive cells express NCAM at a frequency of 21%

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77 Based on this data, I conclude that PYY is primarily expressed in type II taste cells as well as in a small set of type III cells. In this study, I also demonstrated the expression patterns for the Y1 and Y2 receptor. Y1 receptor immunoreactivity display ed obvious cytoplasmic staining allowing us to perform stereological experiments, whereas Y2R staining did not. Quantifying I found that approximately 76% of Y1R positive cells coexpressed 2, ind icating a high percentage of co express ion in type II taste cells. I also determined that 1 1% of cells that express Y1R co express NCAM, indicating a small subset of Y1R positive cells are located in type III taste cells. The expression of Y2R manifested different immunological staining patterns than Y1R. I notice accumulations of positive Y2R reactivity in locations anatomically consistent with the taste pore. This directed our investigations to the possibility of salivary PYY 3 36 interacting with apical Y2 receptors. By manipulating the metabol ic state of the mice, I was able to manipulate levels of PYY in the oral cavity. Basal PYY concentrations are apparent in the saliva of fasted animals and after food intake, these concentrations are increased [84 ]. I determined that the actions of salivar y PYY 3 36 led to an approximately 50% decrease in apical Y2 receptors in the taste bud. Furthermore, the fed mice exhibited an increase in intracellular Y2R immunoreactivity. I attribute this to an increase in internalization of the Y2 receptor, upon activ ation of salivary PYY 3 36 To mimic this system, I acutely augmented salivary PYY 3 36 by oral spray administration and quantified the expression of apically expressed Y2 receptors. Mice that received PYY 3 36 oral spray displayed similar percentages of Y2R expression to the fed group,

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78 also exhibiting an approximately 50% reduction in Y2R expression. Lastly, I report that augmenting NPY in the oral cavity does not affect the expression of apical Y2 receptors. Limitations. There are some caveats surrounding t his data that must be considered. Antibodies made against PYY do not distinguish between PYY 1 36 and PYY 3 36 Due to the absence of DPPIV in taste buds, I suspect that the staining I see in Figure 2 1 is due to PYY 1 36 immunoreactivity; however I e out the option that I am detecting PYY 3 36 diffusing into taste buds via leaky basolateral capillaries. I was unable to quantify cells using the PYY antibody, so I turned to a GFP transgenic model for stereological studies. This yielded GFP positive cel ls present in all of known positive control locations for PYY. Therefore, I assumed that the cells positive for GFP in taste buds are also positive for PYY. However, I was unable to confirm co expression of GFP and PYY in taste cells due to limitations in t he staining protocols. For Y2R staining, I was unable to detect cytoplasmic immunoreactivity. Therefore, I am limited in our capabilities of characterizing its expression pattern by performing stereological experiments. I also visualized varying levels of faint, yet distinct levels of intracellular immunoreactivity for Y2 receptor near the nucleus of taste cells. Two possibilities are assumed based on previously known mechanisms for Y2 receptor expression. First, it could be that I am detecting the interna on ligand interactions. The second possibility is that I am detecting the upregulation of Y2R translation due to activated receptor degradations. Either assumption is equally likely. Future directions. Further immunological st udies should be conducted to determine the expression patterns of the entire NPY system. The expression of NPY,

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79 PP, Y2R, Y4R and Y5R in relation to the taste cell type and to each other can be further in vestigated. Co expression experiments can further divu lge in what subsets of type II cells that PYY and Y1R are expr essed (e.g. T2R or T1R3 ) Also, co expressing PYY with the Y1 receptor would further divulge autocrine vs paracrine signaling in the taste bud. The metabolic impacts on the expression patterns of other Y receptors can be investigated as well. Taste Modulation by PYY S ignaling Chapter 3 of this thesis provides evidence that PYY is modulating taste perception. Using the Davis Rig gustometer to perform brief access taste tests, I compared taste respo nsiveness of PYY / versus wild type mice. These tests are designed to study responses explicit to the oral cavity, reducing potentially confounding physiological variables from peripheral systems. In this study, I demonstrate that PYY / animals display r educed responsiveness for bitter and fat tasting stimuli. I tested two different fat stimuli, corn oil and intralipid emulsions. Entailed in these concoctions is a prosperous amo unt of long chain fatty acids. My data strongly suggest that PYY is effectivel y modulating taste responsiveness for long chain fatty acids. Additionally, PYY / animals showed reduced abilities to respond to two different bitter stimuli, denatonium benzoate and quinine hydrochloride. Therefore, I conclude that PYY signaling in the oral cavity is also modulating bitter responsiveness. Furthermore, no significant differences were observed for sweet, sour or salty stimuli. signaling for these behavioral resp o nses. Using phar macological techniques, I signaling in taste buds of wild type mice, then subjected these mice to brief access

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80 taste testing. By doing so, I observed a reduced r esponse for bitter tasting stimuli similar to the response I observed in PYY / mice. Therefore, I conclude that it is the Y1 administered with BIBO 3304 displayed an in crease in sensitivity to fat stimuli. One thing I can conclude based on this observation is that the Y1 receptor is not mediating candidate (i.e. Y2R). Many other speculati ons surround the reason for this outcome but no definitive conc lusions can be made based on these data alone. To investigate if PYY is acting as an endocrine hormone to impact taste perception, I performed a viral vector experiment. Recombinant adeno asso ciated viral vectors (serotype 5) were packaged coding for either PYY (experimental) or GFP (control) transgenes. Package vectors were administered to the submandibular salivary glands of PYY / mice with no previous PYY signaling present, effectively prod ucing a reconstitution of PYY signaling in the oral cavity of rAAV5 PYY infected mice. I observed that these mice displayed an increase in responsiveness to fat but not bitter tasting stimuli compared to their control (rAAV5 GFP) counterparts. Therefore, I conclude that PYY is acting in an endocrine fashion in the oral cavity to impact fat taste perception. Limitations. Although procedures in the brief access taste test help to minimize the influence of non gustatory influences on the measured behavior I c annot be entirely certain that the impact on responsiveness displayed by PYY / was based on variations in peripheral gustatory functioning As with many behavioral tests using global knockouts, i t is difficult to precisely pinpoint the effects of gene del etion since the gene

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81 is likely expressed in many different tissues In addition, PYY / animals display an upregulation of NPY and the Y2 receptors potentially confounding my data Lastly, i t is known, in some cases, that the Y receptors can impact the dev elopment of tissues. Therefore, for all of these reasons, caution must be used when interpreting our behavior results. All that being said, re sults from both behavioral experi ments that used only WT mice (e.g., BIBO 3304 and rAAV5 PYY) increase our confide nce that the effect observed in our PYY / mice was mediated by the loss of signaling in oral tissues. I am also not certain that BIBO 3304 presented in the oral cavity is able to access the Y1 receptor. Located at the taste pore are claudin rich tight jun ctions. Many molecules are unable to pass through these junctions. However, my data seem to suggest that b ased on our data, it s eems as though the antagonist can efficiently access the Y1 receptor Future directions. Many experiments can be further conduc ted to examine the impact of PYY signaling i n the peripheral gustatory system. Knowing the tastes that are being modulated is only part of the puzzle. Further studies should aim to determine what taste receptors and cell types are mediating the responses f or the stimuli. Further pharmacological studies can be performed to determine the impacts of each Y receptor on taste perception. Patch clamp studies together with calcium imaging, can help to gustatory nerve recordings can conclude if PYY signaling is affecting nerve responses. Theory of PYY Signaling in the Oral C avity In conjunction with previously published data in our lab, I have developed a dynamic, contingent model for PYY signaling in t he oral cavity, depicted in Figure 4 1. Our data suggests two different pools of PYY are present the oral cavity, PYY 1 36 and

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82 PYY 3 36 playing separate roles to impact sepa rate taste behaviors. Acosta et al. 2011 demonstrated reasonable concentration level s of PYY 3 36 in saliva. Their data suggests that PYY 3 36 is diffusing into salivary gland cells via leaky capillaries and is being secreted into saliv a in response to food intake [84 ]. They also determined that Y2 receptors along basal layer of lingual epi thelium are being activated in response to PYY 3 36 signaling. The behavioral outcomes of this activation remain unknown. Howbeit, I do know the behavioral consequences of PYY 3 36 signaling on taste perception. In this study, I found a decrease in apical Y2 R expression levels following food intake, as well as following oral PYY 3 36 augmentation. I attribute this to the activation of Y2 receptors by salivary PYY 3 36 Our behavioral data suggests, salivary PYY 3 36 is impacting fat taste responsiveness. Further more, it is known that PYY 3 36 has a high affinity for the Y2 receptor. Collectively, this data suggests that salivary that PYY 3 36 acts in an endocrine fashion on apically expressed Y2 receptors in the taste bud, ther eby impacting fat taste responsiveness It is interesting to speculate that the anorectic e ffects of oral PYY signaling [84 ] may, in part, be due to its influence on fat responsiveness In brief access taste tests, PYY / mice displayed a reduced res ponse to fat stimuli. This may in part, be due to their inability to properly detect fatty acids requiring them to ingest more fatty foods than mice with normal salivary PYY 3 36 signaling would. Indeed an acute augmentation of oral PYY 3 36 cause s a reduction in food intake [84 ]. Collectively the se behavior al data suggests that increasing PYY signaling in the oral cavity may cause a hypersensitivity to fat stimuli thereby leaving leading to fatty food consumptions. If true, this adds to the on going hypothesis that the gustatory and gastrointestin al systems are parallel in function.

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83 PYY 1 36 comprises our second pool of PYY in the oral cavity. The absence of DDPIV in taste buds prevents its cleavage to PYY 3 36 Also, the presence of apically located tight claudin proteins restricts the pool of PYY 1 36 from departing from the taste bud. In this study, I demonstrated that both PYY 1 36 and Y1Rs are expressed in type II taste cells. While their co expression patterns remain undetermined, I hypothesize that PYY 1 36 is acting in an autocrine fashion on the Y1 recepto r in the taste buds. Herness et al. 2002, demonstrated that the K + rectifying current s (Kir), effectively stabilizing of the resting potential of Y1R positive taste cells. It is known in othe r systems, that PYY 1 36 has a high affinity for the Y1 receptor and results in similar physiological outcomes upon Y1 receptor activation as that observed with NPY Therefore, I hypothesize that PYY 1 36 acting as an autocrine hormone on Y1 receptors in ta ste cells causes an increase in Kir and the stabilization of electrical conductivity. Our pharmacological and behavioral data suggests that this signaling mechanism influences bitter taste perception. The data presented in this thesis posits a dynamic mod el for PYY signaling in the oral cavity. I elaborate on previous findings for the origins of PYY and the Y receptors in taste buds, identifying their exact expression patterns. I distinguish two pools of PYY actin g in separate but distinct roles effective ly impacting taste behaviors I divulge a story of PYY in the oral cavity acting as an endocrine and autcrine hormone to modulate fat and bitter taste perception respectively. Finally, I provide many possible directions for future studies to complete the st ory of PYY and its receptors in the peripheral gustatory system.

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84 Figure 4 1. Theory of PYY Signaling in the Oral Cavity PYY diffuses though leaky capillaries into cells of the salivary glands. Food intake induces s alivary secretions of PYY 3 36 exert ing activity on Y2 receptors of lingual epithelium and apical portions of taste cells Through actions of the Y2 receptor, PYY 3 36 modulates taste cell activity in the presence of long chain fatty acids. With the absence of the DPPIV enzyme in taste buds, PYY 1 36 remains uncleaved PYY 1 36 exerts actions on the Y1 receptor, increasing the activity of rectifying potassium channels and modulation bitter taste perception.

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85 LIST OF REFERENCES 1. Kinnamon SC, Cummings TA (1992) Chemosensory t ransduction mechanisms in taste. Annu Rev Physiol 54: 715 731. 2. Sutherland K, Young RL, Cooper NJ, Horowitz M, Blackshaw LA (2007) Phenotypic characterization of taste cells of the mouse small intestine. Am J Physiol Gastrointest Liver Physiol 292: G142 0 1428. 3. Wu SV, Rozengurt N, Yang M, Young SH, Sinnett Smith J, et al. (2002) Expression of bitter taste receptors of the T2R family in the gastrointestinal tract and enteroendocrine STC 1 cells. Proc Natl Acad Sci U S A 99: 2392 2397. 4. Dyer J, Salmon KS, Zibrik L, Shirazi Beechey SP (2005) Expression of sweet taste receptors of the T1R family in the intestinal tract and enteroendocrine cells. Biochem Soc Trans 33: 302 305. 5. Chen M, Yang Y, Braunstein E, Georgeson KE, Harmon CM (200 1) Gut expression and regulation of FAT/CD36: possible role in fatty acid transport in rat enterocytes. Am J Physiol Endocrinol Metab 281: E916 923. 6. Bezenon C, le Coutre J, Damak S (2007) Taste signaling proteins are coexpressed in solitary intestinal epithelial cells. Chem Senses 32: 41 49. 7. Breer H, Eberle J, Frick C, Haid D, Widmayer P (2012) Gastrointestinal chemosensation: chemosensory cells in the alimentary tract. Histochem Cell Biol 138: 13 24. 8. Laugerette F, Passilly Degrace P, Patris B, Niot I, Febbraio M, et al. (2005) CD36 involvement in orosensory detection of dietary lipids, spontaneous fat preference, and digestive secretions. J Clin Invest 115: 3177 3184. 9. Cartoni C, Yasumatsu K, Ohkuri T, Shigemura N, Yoshida R, et al. (2010) T aste preference for fatty acids is mediated by GPR40 and GPR120. J Neurosci 30: 8376 8382. 10. Degrace Passilly P, Besnard P (2012) CD36 and taste of fat. Curr Opin Clin Nutr Metab Care 15: 107 111. 11. Simons PJ, Kummer JA, Luiken JJ, Boon L (2011) Apic al CD36 immunolocalization in human and porcine taste buds from circumvallate and foliate papillae. Acta Histochem 113: 839 843. 12. Matsumura S, Mizushige T, Yoneda T, Iwanaga T, Tsuzuki S, et al. (2007) GPR expression in the rat taste bud relating to fa tty acid sensing. Biomed Res 28: 49 55.

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94 BIOGRAPHICAL SKETCH Michael Stephen La Sala was born and raised in Ft. Lauderdale, Florida Raised by Robert and Grace La Sala he grew up to develope a strong interest in science. He graduated from Saint Thomas High School and ente red his undergraduate college at Iona College in New Rochelle, New York. After a few years there, he transferred to the was involved in many extra curricular activities in cluding numerous service projects around the Gainesville community Michael entered research as an undergraduate volunteer, working under the that he was thoroughly in degree, Michael join the Interdisciplinary Ph.D program in biomedical sciences, where Related Behavior b t o switch majors and complete a m e. He now aspires to pursue an Medical Doctorate degree and he will be applying to medical school this summer. In the future, Michael wishes t o practice medicine as a medical doctor, while continuing to pursue medically related research